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BimEL up-regulation potentiates AIF translocation and cell death in response to MPTP

Anthony K. F. Liou^*,,1, Zhigang Zhou^*,, Wei Pei^*, Tit-Meng Lim, Xiao-Ming Yin and Jun Chen^*,,

^* Department of Neurology, University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania, USA;
Department of Pathology, University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania, USA;
Pittsburgh Institute for Neurodegenerative Disorders Pittsburgh, Pennsylvania, USA;
Department of Biological Science, National University of Singapore, Singapore; and
Geriatric Research, Educational and Clinical Center Veterans Affairs Pittsburgh Health Care System, Pittsburgh, Pennsylvania, USA

¹Correspondence: Department of Neurology, University of Pittsburgh School of Medicine, S507 Biomedical Science Tower, 3500 Terrace St., Pittsburgh, PA 15261, USA. E-mail: lioukf{at}upmc.edu

SPECIFIC AIMS

The goals of this study were to identify and propose a new caspase independent cell death pathway in response to MPP+.

PRINCIPAL FINDINGS

1. MPP+-induced cell death involved AIF release from the mitochondria
From dosage and time response for cell viability of differentiated PC12 cells to MPP+, we have established that chronic exposure to 5 mM of MPP+ for 24 h elicited 50% cell death reproducibly. Moreover, pretreatment with caspase inhibitors such as z-VAD and DEVD does not attenuate MPP+-induced cell death. Nevertheless, AIF translocation was observed in degenerating cells in response to MPP+. Knocking down the expression of AIF via siRNA effectively attenuated cell death confirming the participation of AIF in this cell death pathway.

2. MPP+-induced BimEL up-regulation that regulates AIF translocation
As shown in Fig.1 A, B, there is an increase in BimEL expression based on Western blot analysis and immunocytochemistry. The temporal profile for BimEL over 24 h showed that the increase in BimEL expression occurred at and after 8 h of chronic exposure to MPP+, which is at the same time point when cell death was detected. Using antisense strategy, we demonstrated that expression of BimEL can be suppressed (Fig. 1C ) and with its suppression, cell death induced by MPP+ is attenuated (Fig. 1D ). Using the same strategy, the suppression of BimEL expression is able to prevent the release from AIF from the mitochondria (Fig. 1E ). This up-regulation of BimEL can be regulated by activated JNK and c-Jun, suggesting that events of JNK and c-Jun activation are chronologically upstream of BimEL up-regulation. In concert, the results suggest that BimEL is another member of the cell death pathway.

View larger version (27K): [in this window] [in a new window]   Figure 1. Up-regulation of BimEL in response to MPP+ treatment. A) Representative Western blots demonstrating the increases in BimEL expression after chronic MPP+ treatment. a) Increased BimEL expression after 24 hours of MPP+ treatment. b) temporal profile of BimEL expression after 0, 0.5, 1, 2, 4, 8, 16, and 24 hours of MPP+ treatment; ß-actin served as a loading control. Fold changes in BimEL expression (derived from 3 independent readings per data point). B) Increased BimEL expression in cells upon MPP+ treatment visualized by immunocytochemistry. a) Image of untreated cells showing virtually no visible BimEL expression. b) Image of cells with visible BimEL expression (green fluorescent) in response to MPP+ treatment for 24 h (cells marked by arrows). C) The efficacy of suppressing BimEL expression by antisense. Suppression of BimEL expression was achieved using the Bim antisense oligonucleotides but not the Bim sense controls, as detected by Western blot analysis with ß-actin as a loading control. D) Suppression of BimEL expression was able to attenuate cell death by approximately 60%. Data are means ± SE, at least 18 readings per data point, from 3 independent experiments. **P < 0.01 vs. Bim sense controls (analysis of variance and post hoc Fisher’s protected least significant difference tests). E) Mitigation of AIF release from the mitochondria to the cytosol after pretreatment with Bim antisense, as detected using Western blot analysis; VDAC and ß-actin served as the loading controls for mitochondrial and cytosolic fractions, respectively. Presented blots are representatives of 2 independent experiments.

3. Calpain activity is increased and involved in cell death in response to MPP+
Besides BimEL expression, MPP+ induced an increase in total calpain activity in differentiated PC12 cells. Subsequent examination has revealed that the expression of calpain I but not calpain II is enhanced in a similar temporal profile as that for the total calpain activity, inferring that the increase in calpain activity may be due to calpain I rather than calpain II. Inhibition of calpain activity via two calpain inhibitors, such as calpeptin and MDL28170 consistently showed an attenuation of cell death induced by MPP+. The inhibition of calpain activity is also able to prevent AIF release from the mitochondria. Together, these results suggest that calpain I can influence AIF release from the mitochondria and cell death.

4. BimEL expression regulates calpain I expression and calpain activity
Since MPP+ induced an increase in BimEL expression as well as calpain activity, we were interested in elucidating the relationship between these two events. As shown in Fig. 2 A, pretreatment with JNK inhibitor SP600125, and calpain inhibitor MDL28170 individually and in combination, attenuated cell death by 50%. The absence of additive protection from these two inhibitors when used in combination suggests that these two events are likely to be part of the same cell death pathway. Moreover, usage of JNK inhibitor is able to suppress BimEL expression but not calpain inhibitor, suggesting that BimEL up-regulation occurs before elevation of calpain activity. To substantiate this finding, we used Bim antisense to knockdown BimEL expression. The suppression of BimEL expression mitigated the increase in calpain activity (Fig. 2C ) and suppressed the increase of calpain I expression (Fig. 2D ). On the whole, the results inferred that the up-regulation of BimEL expression potentiates the elevation in calpain I expression as well as total calpain activity.

View larger version (21K): [in this window] [in a new window]   Figure 2. Suppression of BimEL up-regulation reduces Calpain activation. A) The absence of synergistic or additive attenuation on MPP+-induced cell death by the pretreatment of SP600125 and MDL28170in combination, as compared to the use of either inhibitor alone. Data are means ± SE, at least 12 readings per data point, from 2 independent experiments. *P < 0.05 vs. MPP+ treatment without pretreatment with inhibitors. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference tests. B) Pretreatment with SP600125 but not MDL28170could suppress the up-regulation of BimEL expression as visualized by Western blot analysis. Relative fold changes for BimEL (3 experiments) were presented. C) Suppression of BimEL expression by Bim antisense mitigated the increase in total calpain activity. *P < 0.05 vs. MPP+ treatment alone. Statistics was derived from ANOVA and post hoc Fisher’s protected least significant difference tests (3 experiments). D) Decrease in BimEL expression by Bim antisense could mitigate the increase in the expression of activated calpain I.

CONCLUSIONS AND SIGNIFICANCE

Taken together, the results proposed a novel cell death pathway. The neurotoxin MPP+ elicited the activation of JNK and c-jun, which in turn up-regulated the proapoptotic protein BimEL. The up-regulation of BimEL potentiated the elevation of calpain I expression and total calpain activity which mediated the release of AIF from the mitochondria that lead to cell death.

This is the first study that proposes a caspase independent cell death pathway in response to MPP+. This cell death pathway is consistent with reports demonstrating the importance of calpain, AIF, and JNK activation in MPP+ cell model as well as rodent MPTP model.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3258fje;

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