Limits...
Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis.

Wright KM, Linhoff MW, Potts PR, Deshmukh M - J. Cell Biol. (2004)

Bottom Line: We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell.Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity.Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.

Show MeSH

Related in: MedlinePlus

Modulation of Apaf-1 levels determines the strict IAP regulation of caspases in developing primary sympathetic neurons. (A) Sympathetic neurons from E16 and P3 mice were maintained in culture for 2 d and compared for the ability of cytochrome c to induce apoptosis by cytosolic microinjection of cytochrome c. (B) Levels of Apaf-1 and procaspase-9 were examined in whole-cell lysates of E16 and P3 sympathetic neurons after 2 d in culture. Levels of lactate dehydrogenase (LDH) were examined as a loading control. (C) Primary sympathetic neurons (from P0 mice after 5 d in culture) were injected with plasmid DNAs encoding EGFP (50 ng/μl) and vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c or rhodamine dextran and were assessed for viability at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (D) Model showing how a marked reduction in Apaf-1 activity with neuronal differentiation is coupled to the increased effectiveness of endogenous IAPs in regulating cytochrome c–mediated apoptosis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172554&req=5

fig7: Modulation of Apaf-1 levels determines the strict IAP regulation of caspases in developing primary sympathetic neurons. (A) Sympathetic neurons from E16 and P3 mice were maintained in culture for 2 d and compared for the ability of cytochrome c to induce apoptosis by cytosolic microinjection of cytochrome c. (B) Levels of Apaf-1 and procaspase-9 were examined in whole-cell lysates of E16 and P3 sympathetic neurons after 2 d in culture. Levels of lactate dehydrogenase (LDH) were examined as a loading control. (C) Primary sympathetic neurons (from P0 mice after 5 d in culture) were injected with plasmid DNAs encoding EGFP (50 ng/μl) and vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c or rhodamine dextran and were assessed for viability at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (D) Model showing how a marked reduction in Apaf-1 activity with neuronal differentiation is coupled to the increased effectiveness of endogenous IAPs in regulating cytochrome c–mediated apoptosis.

Mentions: We also examined whether the ability of endogenous IAPs to strictly regulate apoptosis in primary sympathetic neurons (at P5) (Deshmukh et al., 2002; Potts et al., 2003) was a result of decreased Apaf-1 levels with neuronal differentiation. First, we examined sympathetic neurons isolated from embryonic day 16 (E16) mice, a point at which a majority of these neurons have begun postmitotic differentiation and are responsive to NGF (Wyatt and Davies, 1995). After 2 d in culture, these cells were microinjected with cytochrome c and were found to undergo rapid apoptosis with cytochrome c alone (Fig. 7 A). In contrast, P3 sympathetic neurons maintained in culture for 2 d (P5 equivalent) were remarkably resistant to microinjection of cytochrome c because of strict regulation by IAPs, as described previously (Deshmukh and Johnson, 1998; Deshmukh et al., 2002). E16 sympathetic neurons maintained in culture for 8 d (P5 equivalent) also became markedly resistant to cytochrome c (unpublished data).


Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis.

Wright KM, Linhoff MW, Potts PR, Deshmukh M - J. Cell Biol. (2004)

Modulation of Apaf-1 levels determines the strict IAP regulation of caspases in developing primary sympathetic neurons. (A) Sympathetic neurons from E16 and P3 mice were maintained in culture for 2 d and compared for the ability of cytochrome c to induce apoptosis by cytosolic microinjection of cytochrome c. (B) Levels of Apaf-1 and procaspase-9 were examined in whole-cell lysates of E16 and P3 sympathetic neurons after 2 d in culture. Levels of lactate dehydrogenase (LDH) were examined as a loading control. (C) Primary sympathetic neurons (from P0 mice after 5 d in culture) were injected with plasmid DNAs encoding EGFP (50 ng/μl) and vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c or rhodamine dextran and were assessed for viability at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (D) Model showing how a marked reduction in Apaf-1 activity with neuronal differentiation is coupled to the increased effectiveness of endogenous IAPs in regulating cytochrome c–mediated apoptosis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172554&req=5

fig7: Modulation of Apaf-1 levels determines the strict IAP regulation of caspases in developing primary sympathetic neurons. (A) Sympathetic neurons from E16 and P3 mice were maintained in culture for 2 d and compared for the ability of cytochrome c to induce apoptosis by cytosolic microinjection of cytochrome c. (B) Levels of Apaf-1 and procaspase-9 were examined in whole-cell lysates of E16 and P3 sympathetic neurons after 2 d in culture. Levels of lactate dehydrogenase (LDH) were examined as a loading control. (C) Primary sympathetic neurons (from P0 mice after 5 d in culture) were injected with plasmid DNAs encoding EGFP (50 ng/μl) and vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c or rhodamine dextran and were assessed for viability at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (D) Model showing how a marked reduction in Apaf-1 activity with neuronal differentiation is coupled to the increased effectiveness of endogenous IAPs in regulating cytochrome c–mediated apoptosis.
Mentions: We also examined whether the ability of endogenous IAPs to strictly regulate apoptosis in primary sympathetic neurons (at P5) (Deshmukh et al., 2002; Potts et al., 2003) was a result of decreased Apaf-1 levels with neuronal differentiation. First, we examined sympathetic neurons isolated from embryonic day 16 (E16) mice, a point at which a majority of these neurons have begun postmitotic differentiation and are responsive to NGF (Wyatt and Davies, 1995). After 2 d in culture, these cells were microinjected with cytochrome c and were found to undergo rapid apoptosis with cytochrome c alone (Fig. 7 A). In contrast, P3 sympathetic neurons maintained in culture for 2 d (P5 equivalent) were remarkably resistant to microinjection of cytochrome c because of strict regulation by IAPs, as described previously (Deshmukh and Johnson, 1998; Deshmukh et al., 2002). E16 sympathetic neurons maintained in culture for 8 d (P5 equivalent) also became markedly resistant to cytochrome c (unpublished data).

Bottom Line: We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell.Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity.Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.

Show MeSH
Related in: MedlinePlus