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Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

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Proteasome inhibition by MG-132 precedes mitochondria depolarization. (A) Determination of proteasomal activity. N27 dopaminergic neu-ronal cells were treated with 5.0 μM MG-132 for 0-120 min and chymotrypsin-like proteasomal activity was assessed using Suc-LLVY-AMC. Enzymatic activity is presented as percentage of the control group. Values represent mean ± S.E.M for 6 samples in each group. (B) Flow cytometric determination of mitochondrial membrane potential. N27 cells were treated with 5.0 μM MG-132 for 0–120 min. The intensity of red fluorescence for aggregated JC-1 and green fluorescence for monomer JC-1 was determined using flow cytometry, and the red/green ratio was used as the measurement of membrane potential. Values presented as mean ± S.E.M represent results of 4–6 individual samples. (C) Flow cytometric measurements for ROS. N27 cells following exposure to 5.0 μM MG-132 for 0, 20, 40 or 60 min, and intracellular ROS was quantified by flow cytometry using dihy-droethidine fluorescent probe. Data represent the mean ± S.E.M. for 3–5 samples. 200 μM H2O2 was used as positive control. *P<0.05, ***P< 0.001 compared with control group. (D) ROS production using CM-H2DCFDA (DCF) or dihydroethidium (DHE) following 5.0 μM MG-132 treatment for 6 hrs. N27 cells cultured in 96-well plate were treated with 5.0 μM MG-132 for 6 hrs. The cells were then incubated with DCF or DHE before visualization under fluorescence microscopy. H2O2 (50 μM)-treated cells were included as a positive control for ROS production.
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fig01: Proteasome inhibition by MG-132 precedes mitochondria depolarization. (A) Determination of proteasomal activity. N27 dopaminergic neu-ronal cells were treated with 5.0 μM MG-132 for 0-120 min and chymotrypsin-like proteasomal activity was assessed using Suc-LLVY-AMC. Enzymatic activity is presented as percentage of the control group. Values represent mean ± S.E.M for 6 samples in each group. (B) Flow cytometric determination of mitochondrial membrane potential. N27 cells were treated with 5.0 μM MG-132 for 0–120 min. The intensity of red fluorescence for aggregated JC-1 and green fluorescence for monomer JC-1 was determined using flow cytometry, and the red/green ratio was used as the measurement of membrane potential. Values presented as mean ± S.E.M represent results of 4–6 individual samples. (C) Flow cytometric measurements for ROS. N27 cells following exposure to 5.0 μM MG-132 for 0, 20, 40 or 60 min, and intracellular ROS was quantified by flow cytometry using dihy-droethidine fluorescent probe. Data represent the mean ± S.E.M. for 3–5 samples. 200 μM H2O2 was used as positive control. *P<0.05, ***P< 0.001 compared with control group. (D) ROS production using CM-H2DCFDA (DCF) or dihydroethidium (DHE) following 5.0 μM MG-132 treatment for 6 hrs. N27 cells cultured in 96-well plate were treated with 5.0 μM MG-132 for 6 hrs. The cells were then incubated with DCF or DHE before visualization under fluorescence microscopy. H2O2 (50 μM)-treated cells were included as a positive control for ROS production.

Mentions: We used the proteasome inhibitor MG-132 to induce ubiquitin proteasome dysfunction in N27 dopaminergic cells. To systematically examine the effect of the proteasome inhibitor MG-132 on dopaminergic cells, we first performed a detailed time course analysis of chymotrypsin-like proteasomal activity. As shown in Fig.1A, MG-132 exposure led to a rapid inhibition of proteasomal activity, with more than 70% inhibition within 5 min (P< 0.001). Next, we examined whether MG-132 has any effect on mitochondrial membrane potential and ROS generation. Determination of membrane potential by JC-1 showed a gradual and steady reduction starting at 30 min, with a 50% decrease in mitochondrial membrane potential over 120 min of MG-132 (Fig.1B). On the contrary, no significant elevation of ROS was noted during MG-132 treatment (Fig.1C) as measured by flow cytometry. To further confirm ROS production over prolonged exposure to MG-132, fluorescence microscopic analysis were performed. N27 cells were incubated with two different ROS sensitive dyes, dihydroethidine and CM-H2DCFDA, prior to treatment with MG-132 (0.1, 1, 3 or 5 μM) and ROS production was monitored over 6 hrs. As shown in Fig.1D, no significant change in ROS production was detected with either dye in N27 cells exposed to 5 μM MG-132 for 6 hrs. Additionally, lower doses (0.1–3.0 μM) did not generate any ROS (data not shown). However, we observed a significant ROS production in the 50 μM H2O2 treatment (positive control; Fig.1D). These data indicate that proteasomal inhibition, which precedes the dissipation of mitochondria membrane potential, is independent of oxidative insult.


Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Proteasome inhibition by MG-132 precedes mitochondria depolarization. (A) Determination of proteasomal activity. N27 dopaminergic neu-ronal cells were treated with 5.0 μM MG-132 for 0-120 min and chymotrypsin-like proteasomal activity was assessed using Suc-LLVY-AMC. Enzymatic activity is presented as percentage of the control group. Values represent mean ± S.E.M for 6 samples in each group. (B) Flow cytometric determination of mitochondrial membrane potential. N27 cells were treated with 5.0 μM MG-132 for 0–120 min. The intensity of red fluorescence for aggregated JC-1 and green fluorescence for monomer JC-1 was determined using flow cytometry, and the red/green ratio was used as the measurement of membrane potential. Values presented as mean ± S.E.M represent results of 4–6 individual samples. (C) Flow cytometric measurements for ROS. N27 cells following exposure to 5.0 μM MG-132 for 0, 20, 40 or 60 min, and intracellular ROS was quantified by flow cytometry using dihy-droethidine fluorescent probe. Data represent the mean ± S.E.M. for 3–5 samples. 200 μM H2O2 was used as positive control. *P<0.05, ***P< 0.001 compared with control group. (D) ROS production using CM-H2DCFDA (DCF) or dihydroethidium (DHE) following 5.0 μM MG-132 treatment for 6 hrs. N27 cells cultured in 96-well plate were treated with 5.0 μM MG-132 for 6 hrs. The cells were then incubated with DCF or DHE before visualization under fluorescence microscopy. H2O2 (50 μM)-treated cells were included as a positive control for ROS production.
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Related In: Results  -  Collection

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fig01: Proteasome inhibition by MG-132 precedes mitochondria depolarization. (A) Determination of proteasomal activity. N27 dopaminergic neu-ronal cells were treated with 5.0 μM MG-132 for 0-120 min and chymotrypsin-like proteasomal activity was assessed using Suc-LLVY-AMC. Enzymatic activity is presented as percentage of the control group. Values represent mean ± S.E.M for 6 samples in each group. (B) Flow cytometric determination of mitochondrial membrane potential. N27 cells were treated with 5.0 μM MG-132 for 0–120 min. The intensity of red fluorescence for aggregated JC-1 and green fluorescence for monomer JC-1 was determined using flow cytometry, and the red/green ratio was used as the measurement of membrane potential. Values presented as mean ± S.E.M represent results of 4–6 individual samples. (C) Flow cytometric measurements for ROS. N27 cells following exposure to 5.0 μM MG-132 for 0, 20, 40 or 60 min, and intracellular ROS was quantified by flow cytometry using dihy-droethidine fluorescent probe. Data represent the mean ± S.E.M. for 3–5 samples. 200 μM H2O2 was used as positive control. *P<0.05, ***P< 0.001 compared with control group. (D) ROS production using CM-H2DCFDA (DCF) or dihydroethidium (DHE) following 5.0 μM MG-132 treatment for 6 hrs. N27 cells cultured in 96-well plate were treated with 5.0 μM MG-132 for 6 hrs. The cells were then incubated with DCF or DHE before visualization under fluorescence microscopy. H2O2 (50 μM)-treated cells were included as a positive control for ROS production.
Mentions: We used the proteasome inhibitor MG-132 to induce ubiquitin proteasome dysfunction in N27 dopaminergic cells. To systematically examine the effect of the proteasome inhibitor MG-132 on dopaminergic cells, we first performed a detailed time course analysis of chymotrypsin-like proteasomal activity. As shown in Fig.1A, MG-132 exposure led to a rapid inhibition of proteasomal activity, with more than 70% inhibition within 5 min (P< 0.001). Next, we examined whether MG-132 has any effect on mitochondrial membrane potential and ROS generation. Determination of membrane potential by JC-1 showed a gradual and steady reduction starting at 30 min, with a 50% decrease in mitochondrial membrane potential over 120 min of MG-132 (Fig.1B). On the contrary, no significant elevation of ROS was noted during MG-132 treatment (Fig.1C) as measured by flow cytometry. To further confirm ROS production over prolonged exposure to MG-132, fluorescence microscopic analysis were performed. N27 cells were incubated with two different ROS sensitive dyes, dihydroethidine and CM-H2DCFDA, prior to treatment with MG-132 (0.1, 1, 3 or 5 μM) and ROS production was monitored over 6 hrs. As shown in Fig.1D, no significant change in ROS production was detected with either dye in N27 cells exposed to 5 μM MG-132 for 6 hrs. Additionally, lower doses (0.1–3.0 μM) did not generate any ROS (data not shown). However, we observed a significant ROS production in the 50 μM H2O2 treatment (positive control; Fig.1D). These data indicate that proteasomal inhibition, which precedes the dissipation of mitochondria membrane potential, is independent of oxidative insult.

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

Show MeSH
Related in: MedlinePlus