Autophagy is Activated In Vivo during Trimethyltin-Induced Apoptotic Neurodegeneration: A Study in the Rat Hippocampus

Abstract

Trimethyltin (TMT) is an organotin compound known to produce significant and selective neuronal degeneration and reactive astrogliosis in the rodent central nervous system. Autophagy is the main cellular mechanism for degrading and recycling protein aggregates and damaged organelles, which in different stress conditions, such as starvation, generally improves cell survival. Autophagy is documented in several pathologic conditions, including neurodegenerative diseases. This study aimed to investigate the autophagy and apoptosis signaling pathways in hippocampal neurons of TMT-treated (Wistar) rats to explore molecular mechanisms involved in toxicant-induced neuronal injury. The microtubule-associated protein light chain (LC3, autophagosome marker) and sequestosome1 (SQSTM1/p62) (substrate of autophagy-mediated degradation) expressions were examined by Western blotting at different time points after intoxication. The results demonstrate that the LC3 II/I ratio significantly increased at 3 and 5 days, and that p62 levels significantly decreased at 7 and 14 days. Immunofluorescence images of LC3/neuronal nuclear antigen (NeuN) showed numerous strongly positive LC3 neurons throughout the hippocampus at 3 and 5 days. The terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay indicated an increase in apoptotic cells starting from 5 days after treatment. In order to clarify apoptotic pathway, immunofluorescence images of apoptosis-inducing factor (AIF)/NeuN did not show nuclear translocation of AIF in neurons. Increased expression of cleaved Caspase-3 was revealed at 5-14 days in all hippocampal regions by Western blotting and immunohistochemistry analyses. These data clearly demonstrate that TMT intoxication induces a marked increase in both autophagy and caspase-dependent apoptosis, and that autophagy occurring just before apoptosis could have a potential role in neuronal loss in this experimental model of neurodegeneration.

Keywords: Trimethyltin; apoptosis; autophagy; hippocampus; neurodegeneration.

Figure 1

Trimethyltin (TMT) increased LC3II/LC3I, reduced SQSTM1/p62 and Beclin-1 expression levels, and did not affect ATG5 protein in the rat hippocampus after treatment. Graphic presentations and Western blotting images of LC3II/LC3I (a), SQSTM1/p62 (b), Beclin-1 (c), and ATG5 (d) proteins are shown. Values are presented as mean ± standard error of the mean (SEM) for each group: the control rats (n = 3) and rats treated for 3, 5, 7, and 14 days (n = 3/group), * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with controls, Dunnett’s test. Ctrl: control sample.

Figure 2

Localization and expression of LC3 in the rat hippocampal neurons, after TMT treatment, in the CA1 area are shown. Sections of the CA1 field at different post-intoxication time-points (3, 5, 7, and 14 days) are labeled for Neu N (green, a,d,g,j,m), LC3 (red, b,e,h,k,n), and Neu N/LC3 (merge, c,f,i,l,o). Control sections (a–c). At 3 days after treatment, marked LC3 immunoreactivity was observed (e) and LC3 labeling strongly increased at 5 days (h). LC3 staining decreased at 7 (k) and 14 days (n) post intoxication. Note the progressive loss of Neu N-positive neurons in TMT-treated rats (d,g,j,m) compared with control rats (a). Arrows and inserts show details. Scale bar: 100 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Figure 3

Localization and expression of LC3 in the rat hippocampal neurons, after TMT treatment, in the CA3 area are shown. Sections of the CA3 field at different intoxication time-points (3, 5, 7, and 14 days) are labeled for Neu N (green, a,d,g,j,m), LC3 (red, b,e,h,k,n), and Neu N/LC3 (merge, c,f,i,l,o). Control sections (a–c). At 3 days after treatment, marked LC3 immunoreactivity was observed (e) and LC3 labeling strongly increased at 5 days (h). LC3 staining decreased at 7 (k) and 14 days (n) post intoxication. Note the progressive loss of Neu N-positive neurons in TMT-treated rats (d,g,j,m) compared with control rats (a). Arrows and inserts show details. Scale bar: 100 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Figure 4

Localization and expression of LC3 in the rat hippocampal neurons, after TMT treatment, in the CA4 area (#) are shown. Sections of the CA4 field at different intoxication time-points (3, 5, 7, and 14 days) are labeled for Neu N (green, a,d,g,j,m), LC3 (red, b,e,h,k,n), and Neu N/LC3 (merge, c,f,i,l,o). Control sections (a–c). At 3 days after treatment, marked LC3 immunoreactivity was observed (e) and LC3 labeling strongly increased at 5 days (h). LC3 staining decreased at 7 (k) and 14 days (n) post intoxication. Non-relevant LC3 reactivity in the Dentate Gyrus (*) was observed at any time of treatment (e,h,k,n). Note the progressive loss of Neu N-positive neurons in TMT-treated rats (d,g,j,m) compared with control rats (a). Arrows and inserts show details. Scale bar: 100 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Figure 5

An increased number of apoptotic nuclei in the rat hippocampus after TMT treatment is shown. Representative confocal images of the CA1, CA3, and CA4 (#) areas from the hippocampus of control rats (a–c) and rats treated with TMT for 3 (d–f), 5 (g–i), 7 (j–l), and 14 (m–o) days, stained for the presence of apoptotic nuclei (TUNEL staining, green). No apoptotic cells were observed in the Dentate Gyrus (*) at any time of treatment (f,i,l,o). Scale bar: 100 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article. (p) The number of TUNEL-positive nuclei. The number of apoptotic nuclei increased gradually and significantly in all the hippocampal areas starting from 5 up to 14 days post treatment. Values are presented as mean±SEM for each group: the control rats (n = 3) and 3, 5, 7, and 14 days TMT-treated rats (n = 3/group), * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls, Dunnett’s test.

Figure 6

Apoptosis-Inducing Factor (AIF) immunofluorescence labeling in the rat hippocampus after TMT treatment is not localized in the neuron nuclei. Sections of the CA1 field of control (a–c) and different time-point-treated (3, 5, and 7 days, d–i) rats, are labeled for Neu N (green, a,d,g,j), AIF (red, b,e,h,k), and Neu N/AIF (merge, c,f,i,l)). AIF did not translocate to the nucleus and is detected exclusively in the cytoplasm of neurons at 3 (f), 5 (i), and 7 (l) days after treatment. Scale bar: 50 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Figure 7

Increased cleaved Caspase-3 and cytoplasmic Cytochrome c expression levels in the rat hippocampus after TMT treatment. Graphic presentations and Western blotting images of cleaved Caspase-3 (a) and cytoplasmic Cytochrome c (b) proteins are shown. Values are presented as mean±SEM for each group: the control rats (n = 3) and rats TMT-treated for 3, 5, 7, and 14 days (n = 3/group). ** p < 0.01 and *** p < 0.001 compared with controls, Dunnett’s-test. Ctrl: control sample.

Figure 8

Immunohistochemical labeling of cleaved Caspase-3 in the rat hippocampus after TMT treatment is shown. Cleaved Caspase-3 was detected in brown staining (DAB) at 5 and 7 days in some neurons scattered in the CA1 (d,g), CA3 (e,h), and CA4 (# f,i) areas and in numerous neurons at 14 days after treatment in the same regions (respectively j–l). No active Caspase-3 immunoreactivity was observed in the control sections of CA1 (a), CA3 (b), and CA4 (c). No Caspase-3 in the granular neurons of the Dentate Gyrus (*) was observed at any time of treatment (f,i,l). Arrows and inserts show details. Scale bar: 50 µm. Ctrl: control sample, d: days. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.