The altered autophagy mediated by TFEB in animal and cell models of amyotrophic lateral sclerosis

Abstract

Autophagy is an intracellular degradation process that clears away aggregated proteins or aged and damaged organelles. Abnormalities in autophagy result in defects in clearance of these misfolded and aggregate proteins, which have been associated with neurodegenerative disorders. A key neuropathological hallmark of amyotrophic lateral sclerosis (ALS) that contributes to the progressive loss of motor neurons is abnormal protein aggregation of mutant Cu/Zn superoxide dismutase1 (SOD1). TFEB is a recently described gene that regulates autophagy. Several studies have reported that autophagy is altered in ALS, but little is known about the role and mechanisms of TFEB-mediated autophagy during the progression of ALS. In this study, altered expression of TFEB and Beclin-1 were detected in the spinal cords of ALS transgenic mice at different stages and in an NSC-34 cell model with the SOD1-G93A mutation using RT-PCR, western blot, and immunohistochemistry. The majority of cells positive for TFEB and Beclin-1 are β-tubulin III-labeled neurons, especially in the anterior horn of the gray matter. Overexpression of TFEB in NSC-34 cells with the SOD1-G93A mutation increased the mRNA and protein levels of Beclin-1, accompanied by increased levels of LC3-II protein. MTS assay revealed that TFEB overexpression increased proliferation and survival of NSC-34 cells with the SOD1-G93A mutation. Our findings suggest that TFEB promotes autophagy by enhancing the expression of Beclin-1. The altered autophagy mediated by TFEB is a key element in the pathogenesis of ALS, making TFEB a very promising target for the development of novel drugs and new gene therapeutics for ALS.

Keywords: Amyotrophic lateral sclerosis; Beclin-1; NSC-34 cells; SOD1-G93A transgenic mice; TFEB; autophagy.

Figure 1

The expression of TFEB and Beclin-1 in the spinal cords of ALS mice and WT mice. A. Representative RT-PCR of TFEB. β-actin was used as an internal control. B. Representative western blot of TFEB protein. GAPDH was used as an internal control. C. The relative mRNA levels of TFEB as analyzed by RT-PCR (n = 5). D. The relative intensity of TFEB protein as analyzed by western blot (n = 5). E. Representative RT-PCR of Beclin-1. β-actin was used as an internal control. F. Representative western blot of Beclin-1 protein. GAPDH was used as an internal control. G. The relative mRNA level of Beclin-1 as analyzed by RT-PCR (n = 5). H. The relative intensity of Beclin-1 protein as analyzed by western blot (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, vs. WT.

Figure 2

The distribution of TFEB and Beclin-1 in the spinal cords of ALS mice and WT mice detected by immunohistochemical staining. Immunohistochemistry images were taken of gray matter (GM) and white matter (WM) of the spinal cords at different stages. 70 d (A, B, I, J); 95 d (C, D, K, L); 108 d (E, F, M, N); 122 d (G, H, O, P); GM (1); WM (2); WT (A, C, E, G, I, K, M, O); ALS (B, D, F, H, J, L, N, P). Scale bar = 50 μm.

Figure 3

Double immunofluorescence staining results showing the colocalization of TFEB or Beclin-1 with β-tubulin III or GFAP. Representative confocal images were taken of gray matter (GM) of 108-day-old mice. WT (panel 1, 3, 5, and 7) and ALS (panel 2, 4, 6, and 8). Scale bar = 50 μm.

Figure 4

Altered expression of TFEB and Beclin 1 in NSC-34 cells transfected with the pEGFP-SOD1-G93A or pEGFP-SOD1-WT plasmids. A. Representative RT-PCR of TFEB. β-actin was used as an internal control. B. Representative western blot of TFEB protein. GAPDH was used as an internal control. C. The relative mRNA levels of TFEB as analyzed by RT-PCR (n = 5). D. The relative intensity of TFEB protein as analyzed by western blot (n = 5). E. Representative RT-PCR of Beclin-1. β-actin was used as an internal control. F. Representative western blot of Beclin-1 protein. GAPDH was used as an internal control. G. The relative mRNA level of Beclin-1 as analyzed by RT-PCR (n = 5). H. The relative intensity of Beclin-1 protein as analyzed by western blot (n = 5). I. Immunofluorescence staining results showing the expression of TFEB and Beclin-1 in NSC-34 cells 72 h after transfection. Scale bar = 50 μm. **P < 0.01, ***P < 0.001, vs. pEGFP-wt-SOD1.

Figure 5

TFEB overexpression increased the expression of both Beclin-1 and LC3-II and cell survival in NSC-34 cells with a SOD1-G93A mutation. A. Representative RT-PCR of TFEB. β-actin was used as an internal control. B. Representative western blot of TFEB protein. GAPDH was used as an internal control. C. RT-PCR analysis illustrating the overexpression of TFEB (n = 4). D. Western blot analysis illustrating the overexpression of TFEB. The amount of TFEB was quantified and normalized against GAPDH (n = 4). E. Representative RT-PCR of Beclin-1. β-actin was used as an internal control. F. Representative western blot of Beclin-1 protein. GAPDH was used as an internal control. G. RT-PCR analysis illustrating up-regulation of Beclin-1 (n = 4). H. Western blot analysis illustrating the up-regulation of Beclin-1. The amount of Beclin-1was quantified and normalized against GAPDH (n = 4). I. Representative western blot of LC3-II protein. GAPDH was used as an internal control. J. Western blot analysis illustrating up-regulation of LC3-II. The amount of LC3-II was quantified and normalized against GAPDH (n = 4). K. TFEB overexpression increases cell survival of NSC-34 cells with the SOD1-G93A mutation evaluated by MTS assay. *P < 0.05, **P < 0.01, ***P < 0.001, vs. DsRed2 vector.