Autophagy alleviates neurodegeneration caused by mild impairment of oxidative metabolism

Abstract

Thiamine deficiency (TD) causes mild impairment of oxidative metabolism and region-selective neuronal loss in the brain, which may be mediated by neuronal oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation. TD-induced brain damage is used to model neurodegenerative disorders, and the mechanism for the neuronal death is still unclear. We hypothesized that autophagy might be activated in the TD brain and play a protective role in TD-induced neuronal death. Our results demonstrated that TD induced the accumulation of autophagosomes in thalamic neurons measured by transmission electron microscopy, and the up-regulation of autophagic markers LC3-II, Atg5, and Beclin1 as measured with western blotting. TD also increased the expression of autophagic markers and induced LC3 puncta in SH-SY5Y neuroblastoma cells. TD-induced expression of autophagic markers was reversed once thiamine was re-administered. Both inhibition of autophagy by wortmannin and Beclin1 siRNA potentiated TD-induced death of SH-SY5Y cells. In contrast, activation of autophagy by rapamycin alleviated cell death induced by TD. Intraperitoneal injection of rapamycin stimulated neuronal autophagy and attenuated TD-induced neuronal death and microglia activation in the submedial thalamus nucleus (SmTN). TD inhibited the phosphorylation of p70S6 kinase, suggesting mTOR/p70S6 kinase pathway was involved in the TD-induced autophagy. These results suggest that autophagy is neuroprotective in response to TD-induced neuronal death in the central nervous system. This opens a potential therapeutic avenue for neurodegenerative diseases caused by mild impairment of oxidative metabolism. Autophagy is neuroprotective in response to thiamine deficiency (TD)-induced neuronal death. TD caused neuronal damage and induced the formation of autophagosome, and increased the expression of autophagy-related proteins. Autophagy sequestered damaged and dysfunctional organelles/protein, and transported them to lysosomes for degradation/recycling. This process provided nutrients for injured neurons. Wortmannin and knockdown of Beclin1 inhibited autophagy, and exacerbated TD-induced cell death, while activation of autophagy by rapamycin offered protection against TD neurotoxicity.

Keywords: autophagy; neurodegeneration; oxidative stress; thalamus; vitamin B1.

Fig. 1

Thiamine deficiency (TD)-induced autophagy in the thalamus. (A) TD increased the expression of LC3-II and Beclin1 in the thalamus as determined by immunoblotting. (B) The relative amount of LC3-II and Beclin1 was quantified and normalized to the expression of GAPDH. The results were mean ± SEM (n = 5, *p < 0.05, compared with control group). (C) TD increases the immunoreactivity of LC3 and Beclin1 (green) in NeuN-positive neurons (red) in SmTN after TD for 9 days. Scale bar = 10 µm. (D) The autophagosomes within neurons in SmTN were revealed by electron microscopy (arrows) after TD for 10 days. Scale bar = 500 nm.

Fig. 2

TD-induced autophagy in SH-SY5Y neuroblastoma cells. (A) The expression of LC3, phosphorylated p70S6K (p-P70), p70S6K (t-P70), Atg5 and Beclin1 increased after TD for the indicated times as determined by immunoblotting; TD-induced alterations were reversed by re-administration of thiamine. (B) The relative amount of LC3-II, Atg5 and Beclin1 was quantified and normalized to the expression of GAPDH. p-P70 was quantified and normalized to the expression of t-P70. The results were means ± SEM of three replicates (*p < 0.05, compared with Ct; #p < 0.05, compared with TD7). (C) GFP-LC3 puncta were revealed by confocal microscopy for the indicated times. Scale bar = 5 µm. (D) GFP-LC3 puncta per cell were determined as described under the Materials and Methods. The results were means ± SEM of three replicates (*p < 0.05, compared with Ct; #p < 0.05, compared with TD7). (E) Leupeptin and pepstatin A (LP) increased the level of LC3-II, but had no effect on p-P70. (F) The relative amount of LC3 II was quantified and normalized to GAPDH. p-P70 was quantified and normalized to t-P70. The results were means ± SEM of three replicates (*p < 0.05, compared with controls; #p < 0.05, compared with TD5 or TD7 without LP).

Fig. 3

Effect of wortmannin on TD-induced autophagy and cell death. SH-SY5Y cells were treated with pyrithiamine (0 or 300 nmol/L) and wortmannin (Wort, 0 or 50 nM) for 5 days. (A) Wortmannin decreased TD-induced upregulation of LC3-II, Beclin1 and Atg5. (B) The expression of LC3-II, Beclin1 and Atg5 was quantified and normalized to GAPDH. (C) Wortmannin decreased TD-induced upregulation of GFP-LC3 puncta. Scale bar = 5 µm. (D) The GFP-LC3 puncta per cell were counted. (E) Inhibition of autophagy by wortmannin promoted TD-induced cell death was determined by MTT assay. (F) The cell death was measured by Annexin-V/PI staining. All of the results were means ± SEM of three replicates (*p < 0.05, compared with Ct without wortmannin; #p < 0.05, compared with TD without wortmannin).

Fig. 4

Effect of Beclin1 siRNA on TD-induced autophagy and cell death. (A) Beclin1 siRNA decreased TD-induced increase of LC3-II and Beclin1. (B) The relative amount of LC3-II and Beclin1 was quantified and normalized to GAPDH. (C) Beclin1 siRNA decreased TD-induced increase of GFP-LC3 puncta. Scale bar = 5 µm. (D) The GFP-LC3 puncta per cell were quantified. (E and F) Beclin1 siRNA exacerbated TD-induced cell death was determined by MTT assay (E) and Annexin-V/PI staining (F). These results were means ± SEM of three replicates (*p < 0.05, compared with Ct with siControl; #p < 0.05, compared with TD with siControl).

Fig. 5

Effect of rapamycin on TD-induced autophagy and cell death. SH-SY5Y cells were treated with pyrithiamine (0 or 300 nmol/L) and rapamycin (Rapa, 0 or 10 nM) for 5 days. (A) The expression of LC3, p-P70, and t-P70 was determined by immunoblotting. (B) The relative amount of LC3-II was quantified and normalized to GAPDH. p-P70 was quantified and normalized to t-P70. (C) Rapamycin increases TD-induced upregulation of GFP-LC3 puncta in SH-SY5Y cells. Scale bar = 5 µm. (D) The GFP-LC3 puncta per cell were quantified. (E and F) Rapamycin protected TD-induced cell death measured by the cell viability (E) and Annexin-V/PI staining (F). All the results were means ± SEM of three replicates (*p < 0.05, compared with Ct without rapamycin; #p < 0.05, compared with TD without rapamycin).

Fig. 6

Effect of rapamycin on TD-induced neuronal death and microglia activation in SmTN. (A) Rapamycin induced LC3-positive (green) puncta increased in NeuN-positive (red) cells in SmTN. Scale bar = 10µm. (B) TD-induced loss of NeuN-positive cells in SmTN was reduced by the treatment with rapamycin. Left panel: NeuN immunohistochemistry (IHC) was performed to identify neurons in SmTN. Scale bar = 50 µm. Right panel: NeuN-positive cells in SmTN were quantified by stereological analysis. (C) TD-induced IbaI-positive microglia activation in SmTN was reduced by the treatment with rapamycin. Left panel: IbaI IHC was performed to identify active microglia in SmTN. Scale bar = 50 µm. Right panel: The IbaI immunoreactivity in SmTN was quantified. (D) TD-induced astrocyte activation in SmTN was reduced by the treatment with rapamycin. Left panel: GFAP IHC was performed to identify astrocytes in SmTN. Scale bar = 50 µm. Right panel: The GFAP immunoreactivity in SmTN was quantified. All the results were mean ± SEM of five animals (*p < 0.05, compared with Ct without rapamycin; #p < 0.05, compared with TD without rapamycin).