Prostate Apoptosis Response-4 Enhances Secretion of Amyloid beta  Peptide 1-42 in Human Neuroblastoma IMR-32 Cells by a Caspase-dependent Pathway*

Qing GuoDagger, Jun Xie, Xiaowei Chang, and Huimin Du

From the Department of Neurobiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095

Received for publication, December 6, 2000, and in revised form, February 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Prostate apoptosis response-4 (Par-4) is a leucine zipper protein that promotes neuronal cell death in Alzheimer's disease (AD). Neuronal degeneration in AD may result from extracellular accumulation of amyloid beta  peptide (Abeta ) 1-42. To examine the effect of Par-4 on Abeta secretion and to reconcile amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that overexpress Par-4 and/or its leucine zipper domain. Overexpression of Par-4 did not significantly affect levels of the endogenously expressed beta  amyloid precursor protein but drastically increased the Abeta 1-42/Abeta total ratio in the conditioned media about 6-8 h after trophic factor withdrawal. Time course analysis of caspase activation reveals that Par-4 overexpression exacerbated caspase activation, which is detectable within 2 h after trophic factor withdrawal. Furthermore, inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. In addition, the effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the leucine zipper domain, indicating that the effect of Par-4 on Abeta secretion may require its interaction with other protein(s). These results suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mutations in familial Alzheimer's disease genes, such as beta -amyloid precursor protein (APP)1, presenilin-1, and presenilin-2, have been shown to regulate the processing of APP and result in increased production of the longer form of amyloid beta  peptide (Abeta ), Abeta 1-42 (1-5). It is widely accepted that neuronal degeneration in AD is caused by extracellular accumulation of Abeta 1-42. On the other hand, all three familial Alzheimer's disease genes have been shown to regulate neuronal apoptosis, suggesting that dysregulation of apoptotic pathways may play an important role in neuronal degeneration in AD (6-10). Importantly, abnormal processing of APP and increased production of Abeta may be induced by apoptotic insults (11-14). Several studies show that APP in neuronal cells can be processed by caspase-6 and -8 and that this processing can be blocked by caspase inhibitors (12, 15).

We recently identified Par-4 as a novel cell death-promoting protein associated with neuronal degeneration in AD (16, 17). The pro-apoptotic actions of Par-4 require its interaction with other proteins via the leucine zipper domain (Leu.zip; Refs. 16 and 17). Overexpression of Leu.zip of Par-4 has been used as a dominant negative regulator of Par-4 function because it competitively blocks the interaction of Par-4 with other proteins, thereby blocking the pro-apoptotic actions of Par-4. Blockade of Par-4 function by Par-4 antisense treatment or overexpression of Leu.zip significantly decreases neuronal apoptosis induced by Abeta , trophic factor withdrawal, or overexpression of Alzheimer's mutant presenilin-1 proteins in PC12 cells (16). In recent studies in mutant presenilin-1 M146V knock-in (PS1mv KI) transgenic mice (18-20), we found that the Alzheimer's presenilin-1 mutation enhances Par-4 expression, resulting in increased vulnerability of neurons to apoptosis and increased production of Abeta 1-42 (18-20). These data strongly suggest that induction of Par-4 is an important and necessary event in the pathogenic mechanisms of Alzheimer's presenilin-1 mutations and is very likely involved in the abnormal processing of APP during the apoptotic process. To provide better solutions for the treatment of Alzheimer's disease, it is very important to reconcile amyloid/apoptosis hypotheses of AD and examine how Par-4 might alter APP processing during apoptosis, leading to increased production of the neurotoxic Abeta 1-42.

The cell types responsible for overproduction of Abeta 1-42 are not completely known, although it has been suggested that Abeta 1-42 is produced by human central nervous system neurons from APP695 (1-5). Human neuroblastoma IMR-32 cells have many features that resemble human neurons in the central nervous system (21-23). Importantly, compared with many other human neuronal cell lines, IMR-32 cells have been shown to be able to synthesize a large amount of endogenous APP751 and APP695 and secrete a significant amount Abeta 1-42 together with Abeta 1-40 (21-23). In addition, Abeta secreted by IMR-32 cells can be effectively detected using highly sensitive sandwich ELISAs (21-23). These findings suggest that IMR-32 cells may provide a unique system to examine the possible role of Par-4 in APP processing. We report in this study that overexpression Par-4 increases secretion of Abeta 1-42 from IMR-32 cells after trophic factor withdrawal by a mechanism involving interaction of Par-4 with other proteins via the leucine zipper domain. In addition, we provide evidence showing that the increase in secretion of Abeta 1-42 induced by Par-4 is a caspase-dependent event and occurs when apoptotic cascades are initiated.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation and Characterization of IMR-32 Cell Lines and Trophic Factor Withdrawal-- Human neuroblastoma IMR-32 cells (American Type Culture Collection) were maintained at 37 °C in an atmosphere of 95% air and 5% CO2 in Eagle's minimum essential medium supplemented with nonessential amino acids and 10% heat-inactivated fetal bovine serum. To express the full-length Par-4 in IMR-32 cells, we subcloned a full-length Par-4 cDNA into the expression vector pRc/CMV, yielding a recombinant construct pCMV-Par4, in which the expression of the transgene was driven by enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus. This construct encodes a 1.2-kilobase RNA species and a full-length 38-kDa Par-4 protein. To address the role of the leucine zipper domain of Par-4 in IMR-32 cells, we subcloned the cDNA of the leucine zipper domain of Par-4 into the pREP4 expression vector, where the expression of the transgene is under the control of the Rous sarcoma virus long terminal repeat promoter. pREP-Leu.zip encodes an ~400-base pair RNA species and the ~10-kDa leucine zipper domain of Par-4 protein. IMR-32 cells were transfected with pCMV-Par4 or pREP-Leu.zip or co-transfected with pCMV-Par4 and pREP-Leu.zip using LipofectAMINE (Life Technologies, Inc.), and stable transfectants were selected for 4 weeks using G418 (for pCMV-Par4-transfected cells) and/or hygromycin (for pREP-Leu.zip-transfected cells). Overexpression of Par-4 and/or Leu.zip in IMR-32 cells did not significantly affect the viability of these cells under basal conditions. Selected clones of transfected cells were maintained in the culture medium containing G418 (400 µg/ml; for pCMV-Par4-transfected cells) or hygromycin (400 µg/ml; for pREP-Leu.zip-transfected cells) or both for co-transfected cells. For control purposes, parallel cultures of IMR-32 cells were stably transfected with the pRc/CMV and pREP4 vectors alone. After the cells became confluent in the culture flasks, the culture medium was replaced with fresh media, and cells were incubated for 48 h at 37 °C to condition the medium for Abeta measurement. Trophic factor withdrawal was initiated by washing cultures four times with Locke's buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, and 5 mM HEPES, pH 7.2) with subsequent incubation in 1 ml of Locke's buffer.

Western Blot Analysis-- Levels of Par-4 in cultured IMR-32 cells were determined by Western blot analysis as described in our previous studies (16). Western blot images were acquired and quantified using Kodak Image Station 440 CF and Kodak Digital Science 1D 3.0.2. software. Generation and characterization of the affinity-purified anti-Par-4 antibody have been described previously (16). The total amount of cellular APP in various transfected cell lines was measured by Western blot analysis using a monoclonal antibody (22C11; Roche Molecular Biochemicals) that recognizes the N terminus of human APP. Equal loading was verified by probing with the anti-alpha -tubulin antibody (Sigma).

Quantification of Levels of Abeta 1-40 and Abeta 1-42 by Sandwich ELISAs-- Abeta 1-40 and Abeta 1-42 levels in the conditioned media were measured using a fluorescence-based sandwich ELISA described previously (19). The C-terminal-specific sandwich ELISAs use a monoclonal antibody directed against the N-terminal region of human Abeta and two other antibodies specific for Abeta 1-40 and Abeta 1-42 (BIOSOURCE). Conditioned culture media were collected and subjected to the sandwich ELISAs according to the manufacturer's instructions. Standard curves were generated using lyophilized synthetic human Abeta 40 and human Abeta 42 standards. The standard Abeta 40 and Abeta 42 peptides were chemically modified by the manufacturer to reduce aggregation of the peptides. Parallelism between the standard Abeta peptides used in this study and the native Abeta 40 and Abeta 42 peptides in terms of their immunoreactivity and reliability in the ELISA has been well characterized by the manufacturer. The specificity and sensitivity of the ELISAs have also been characterized by the manufacturer. Fluorescence was quantified with a fluorescence plate reader with excitation at 460 nm and emission at 560 nm. Because absolute values of Abeta 1-40 and Abeta 1-42 secreted by IMR-32 cells under basal culture conditions showed some variability among and even within cell lines, the ratio (percentage) of Abeta 1-42 to the total amount of Abeta (Abeta 1-40 plus Abeta 1-42), which remains statistically consistent under basal culture conditions among the various cell lines, was used to measure the changes in the relative amount of Abeta 1-42 secreted from transfected IMR-32 cells.

Assessments of Apoptosis and Quantification of Caspase-3 Activity-- Apoptosis was quantified in cultures stained with the fluorescent DNA-binding dye Hoechst 33342 as described previously (16). Hoescht 33342-stained cells were visualized and photographed under epifluorescence illumination (340 nm excitation and 510 nm barrier filter) using a ×40 oil immersion objective (200 cells per culture were counted, and counts were made in at least six separate cultures per treatment condition). Samples were analyzed without knowledge of the treatment history of the cultures. Levels of caspase-3 activity were quantified using a method that employed DEVD, a pseudosubstrate and inhibitor of caspase-3 (16). After treatment, cells were incubated for 20 min in the presence of 0.02% digitonin plus 10 µg/ml biotinylated DEVD-CHO (Calbiochem, La Jolla, CA), washed three times with phosphate-buffered saline (2 ml/wash), and fixed for 30 min in a cold solution of 4% paraformaldehyde in phosphate-buffered saline. Cells were then incubated for 5 min in phosphate-buffered saline containing 0.2% Triton X-100, followed by a 30-min incubation in phosphate-buffered saline containing 5 µg/ml Oregon Green-streptavidin (Molecular Probes, Eugene, OR). Confocal images of cellular fluorescence (corresponding to conjugates of activated caspase-3 with DEVD-biotin) were acquired (488 nm excitation and 510 nm emission). All images were acquired using the same laser intensity, offset, and electronic gain to allow quantitative comparisons of relative levels of fluorescence in cells. The average pixel intensity/cell body was determined using Fluoview 2.0 software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation and Characterization of IMR-32 Cell Lines Overexpressing Full-length Par-4 and/or the Leucine Zipper Domain-- To establish the role of Par-4 in Abeta secretion and its possible relationship to apoptotic cell death and to address the ability of the leucine zipper domain to mediate Par-4 actions in neural cells, we generated IMR-32 cell lines overexpressing full-length Par-4, Leu.zip alone, or a combination of full-length Par-4 and Leu.zip. In previous studies, we established that the leucine zipper domain fragment of Par-4 acts as a dominant negative regulator of Par-4 activity when expressed in neural cells, possibly by competitively abrogating the binding of Par-4 to other proteins (16). On Western blots, the Par-4 polyclonal antibody recognized full-length Par-4 (38 kDa) and Leu.zip (10 kDa) (Fig. 1). We chose for study two to three clones of each of the cells overexpressing full-length Par-4, Leu.zip, and Par-4+Leu.zip, based on their similar levels of expression of Par-4 and/or Leu.zip (Fig. 1). Western blot analyses showed that levels of full-length Par-4 protein were ~8-fold higher in cell lines transfected with Par-4 cDNA compared with untransfected, vector-transfected, and Leu.zip-transfected cell lines. Bands of the leucine zipper domain of Par-4 were clearly identified in cells transfected or co-transfected with Leu.zip cDNA. Note that overexpression of Par-4 and/or Leu.zip did not significantly affect the levels of the endogenously expressed APP in the various cell lines used in this study.


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Fig. 1.   Representative Western blots showing levels of expression of Par-4 and the leucine zipper domain in transfected IMR-32 cells used in the present study. Proteins from the indicated cell lines were separated by SDS-polyacrylamide gel electrophoresis (50 µg protein/lane), transferred to a nitrocellulose sheet, and immunoreacted with a polyclonal anti-Par-4 antibody (top), a monoclonal antibody that recognizes the N terminus of human APP (middle), or a monoclonal anti-alpha -tubulin antibody (bottom). Untransfected, untransfected parent cell line; Vector alone, cell line transfected with empty vector; Par-4, C9, Par-4, C6, and Par-4, C3, cell lines overexpressing full-length Par-4; Par4+Leu.zip, C1, Par4+Leu.zip, C3, and Par4+Leu.zip, C12, cell lines overexpressing both Par-4 and Leu.zip; Leu.zip, C8 and Leu.zip, C10, cell lines overexpressing Leu.zip. Note that transfected cell lines that overexpress Par-4 express comparable levels of full-length Par-4 and that transfected cell lines that overexpress Leu.zip express comparable levels of Leu.zip. The total amount of cellular APP was at similar levels among the various transfected cell lines.

Effect of Overexpression of Par-4 on Secretion of Abeta 1-42 from IMR-32 Cells under Basal Culture Conditions and after Trophic Factor Withdrawal-- Overexpression of Par-4 did not result in a significant increase in spontaneous apoptosis in IMR-32 cells. To examine whether overexpression of Par-4 and/or Leu.zip alters Abeta secretion from IMR-32 cells under basal conditions, levels of Abeta 1-40 and Abeta 1-42 in the conditioned media of various cell lines were measured using a highly sensitive fluorescence-based sandwich ELISA. As shown in Fig. 2, values of the Abeta 1-42/Abeta total ratio in the conditioned culture media of wild type and vector-transfected control IMR-32 cells were about 11-12%. Overexpression of Par-4, overexpression of Leu.zip, or co-expression of Par-4 and Leu.zip did not result in significant changes in the values of the Abeta 1-42/Abeta total ratio in the conditioned culture media of various transfected IMR-32 cell lines, indicating that Par-4 does not alter APP processing under basal culture conditions. Because Par-4 increases the vulnerability of neuronal cells to apoptosis induced by trophic factor withdrawal and Abeta (16), and because APP has been reported to be processed in neuronal cells during apoptosis by some cell death proteases (12, 15), we sought to examine whether overexpression of Par-4 alters Abeta secretion from IMR-32 cells after exposure of the cells to apoptotic insults, such as trophic factor withdrawal. Time course analyses of secretion of Abeta peptides in IMR-32 cells showed that values of the Abeta 1-42/Abeta total ratio in untransfected and vector-transfected control cells and in cells overexpressing Leu.zip remained statistically unchanged for up to 8 h after trophic factor withdrawal. This indicates that trophic factor withdrawal itself is not sufficient to induce a significant increase in secretion of Abeta 1-42 in IMR-32 cells (Fig. 3). However, overexpression of Par-4 drastically increased the Abeta 1-42/Abeta total ratio in the conditioned media about 6-8 h after trophic factor withdrawal (Fig. 3). Consistent with a dominant negative mode of action of Leu.zip involving protein-protein interactions, co-overexpression of Leu.zip completely abolished the adverse effect of Par-4 on Abeta secretion, indicating that actions of Par-4 in Abeta secretion require its interaction with other protein(s) via the leucine zipper domain (Fig. 3).


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Fig. 2.   Under basal culture conditions, overexpression of Par-4 does not alter the relative amount of Abeta 1-42 secreted by IMR-32 cells. The percentage of Abeta 1-42 as a fraction of total Abeta (Abeta 1-40 + Abeta 1-42) secreted under basal culture conditions by the indicated clones of transfected PC12 cells was quantitated by sandwich ELISAs as detailed under "Materials and Methods." Untransfected, untransfected parent cell line; Vector Alone, cell line transfected with empty vector; Par-4, C9, cell line overexpressing full-length Par-4; Leu.zip, C8, cell line overexpressing the Leu.zip of Par-4; Par4+Leu.zip, C3, cell line that overexpresses both full-length Par-4 and the Leu.zip of Par-4. Values are the mean and S.E. of determinations made in six separate cultures. ***, p < 0.001 compared with values of the Abeta 1-42/Abeta total ratio in untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post hoc tests.


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Fig. 3.   Par-4 significantly increases secretion of Abeta 1-42 from transfected IMR-32 cells after trophic factor withdrawal: complete blockade by co-overexpression of Leu.zip. Cultures of the indicated clones of transfected IMR-32 cells were deprived of trophic support for the indicated time periods, and values of the Abeta 1-42/Abeta total ratio in the conditioned culture media of transfected IMR-32 cells were measured by sandwich ELISAs. Note that the values of the Abeta 1-42/Abeta total ratio in untransfected and vector-transfected control cells and in cells overexpressing Leu.zip remained statistically unchanged after trophic factor withdrawal. However, overexpression of Par-4 drastically increased the Abeta 1-42/Abeta total ratio in the conditioned media. This effect of Par-4 was not observed until ~6 h after trophic factor withdrawal. Co-overexpression of Leu.zip completely abolished the adverse effect of Par-4 on Abeta secretion. Values are the mean and S.E. of determinations made in six separate cultures. ***, p < 0.001 compared with the corresponding values of the Abeta 1-42/Abeta total ratio in untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post hoc tests.

Par-4 Increases Secretion of Abeta 1-42 after Initiation of Apoptotic Cascades and by a Caspase-dependent Pathway-- To test the hypothesis that Par-4 increases Abeta 1-42 secretion by initiating early apoptotic cascades, we performed additional experiments to examine whether Par-4 increases the vulnerability of IMR-32 cells to apoptosis induced by trophic factor withdrawal. Fig. 4 shows the impact of overexpression of Par-4 on apoptosis of IMR-32 cells. Each of the cell lines was deprived of trophic support for up to 48 h, and cells with apoptotic nuclei were counted. Basal levels of apoptosis ranged from 2-5% among the various cell lines. Forty-eight h after trophic factor withdrawal, apoptosis was found in 33-42% of the control cell lines and in cells overexpressing Leu.zip. Levels of trophic factor withdrawal-induced apoptosis were significantly increased to about 74% in cells overexpressing Par-4. Co-overexpression of Leu.zip completely blocked trophic factor withdrawal-induced apoptosis and the pro-apoptotic actions of Par-4. These results clearly demonstrate that Par-4 does promote apoptosis in IMR-32 cells after trophic factor withdrawal. Because cysteine proteases of the caspase family have a prominent role in apoptosis, we examined the levels of caspase-3 activity and determined whether Par-4 played a role in caspase activation in IMR-32 cells after trophic factor withdrawal. As shown in Fig. 5, trophic factor withdrawal-induced caspase activation was exacerbated in cells overexpressing Par-4, and Leu.zip suppressed caspase activation induced by Par-4, indicating an important role for Par-4 in IMR-32 cells in the early period of the apoptotic cascades before caspase activation. To determine whether the exacerbated caspase activation was responsible for the increase in Abeta 1-42 secretion after trophic factor withdrawal, we examined whether the broad spectrum caspase inhibitor BD-fmk could block the Par-4-induced increase in Abeta 1-42 secreted by IMR-32 cells. As shown in Fig. 6, the Par-4-induced increase in secretion of Abeta 1-42 after trophic factor withdrawal was significantly attenuated by inhibition of caspase activation. Thus, 8 h after trophic factor withdrawal, the Abeta 1-42/Abeta total ratio in the conditioned medium of cells overexpressing Par-4 was reduced from 57% without BD-fmk treatment to 23% with BD-fmk treatment. These results strongly suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway.


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Fig. 4.   Par-4 increases the vulnerability of IMR-3 cells to apoptosis induced by trophic factor withdrawal: protection by co-overexpression of Leu.zip. Cultures of the indicated clones of transfected IMR-32 cells were deprived of trophic support for the indicated time periods, and the percentage of cells with apoptotic nuclei was then quantified. Values are the mean and S.E. of determinations made in six separate cultures (measurements were made in 300-600 cells/culture). The cell lines used were the same as those described in the Fig. 1 legend. ***, p < 0.01 compared with the corresponding values for untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post hoc tests.


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Fig. 5.   Early and exacerbated activation of caspase activity in cells overexpressing Par-4 after trophic factor withdrawal. Cultures of the indicated clones of transfected IMR-32 cells were deprived of trophic support for the indicated time periods, and levels of cellular DEVD fluorescence, a measure of caspase-3 activity, were quantified. Note that trophic factor withdrawal-induced caspase activation was significantly exacerbated in cells overexpressing Par-4 as early as 2 h after trophic factor withdrawal. Co-overexpression of Leu.zip completely blocked the effect of Par-4 on caspase activation. Values are the mean and S.E. of determinations made in six separate cultures. ***, p < 0.001 compared with corresponding values in untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. Similar data were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post hoc tests.


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Fig. 6.   The broad spectrum caspase inhibitor BD-fmk counteracts the adverse effect of Par-4 on APP processing after trophic factor withdrawal. Cultures of the indicated clones of transfected IMR-32 cells were pretreated with 100 µM of the broad spectrum caspase inhibitor BD-fmk (Enzyme Systems Products, Livermore, CA) for 2 h and then deprived of trophic support for 8 h. Parallel cultures were prepared and deprived of trophic support for 8 h without BD-fmk pretreatment. Values of the Abeta 1-42/Abeta total ratio in the conditioned culture media of transfected IMR-32 cells were measured by sandwich ELISAs. Note that overexpression of Par-4 drastically increased the Abeta 1-42/Abeta total ratio. Inhibition of caspase inhibition by BD-fmk significantly inhibited the adverse effect of Par-4 on Abeta 1-42 secretion. Values are the mean and S.E. of determinations made in six separate cultures. ***, p < 0.001 compared with corresponding values of the Abeta 1-42/Abeta total ratio in untransfected, vector-transfected, Leu.zip, and Par4+Leu.zip cell groups. ###, p < 0.001 compared with the value of the Abeta 1-42/Abeta total ratio in Par-4 cells without BD-fmk pretreatment. Similar data were obtained from cell lines Par-4, C6 and Par-4, C3; Leu.zip, C10; and Par4+Leu.zip, C1 and Par4+Leu.zip, C12. ANOVA was performed with Scheffe's post hoc tests.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To examine the possible effect of Par-4 on APP processing, we generated and characterized stable transfected IMR-32 cell lines that overexpress Par-4 and/or Leu.zip. Notably, overexpression of Par-4 and/or Leu.zip did not significantly affect the levels of the endogenously expressed APP in the various cell lines used in this study. We found that Par-4 increases secretion of Abeta 1-42 from IMR-32 cells after trophic factor withdrawal. We further addressed some key questions about the mechanisms by which Par-4 causes the observed increase in Abeta 1-42 production. We found that Par-4 induced an early and exacerbated activation of caspase-3 activity that preceded the increase in Abeta 1-42 secreted by IMR-32 cells. Inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. These results strongly suggest that Par-4 is a novel mediator of aberrant APP processing and that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.

Overexpression of Par-4 did not result in a significant increase in spontaneous apoptosis in IMR-32 cells, indicating that Par-4 overexpression alone is not sufficient to induce apoptosis. These findings are consistent with those of our previous study of cultured prostate tumor cells and PC12 cells, in which overexpression of Par-4 in transiently or stably transfected cells did not induce apoptosis (16, 17). In addition, overexpression of Par-4 or Leu.zip or co-expression of Par-4 and Leu.zip did not result in significant changes in the values of the Abeta 1-42/Abeta total ratio in the conditioned medium of various transfected IMR-32 cell lines under basal culture conditions. This result is in sharp contrast to the reported effect of mutant presenilins, where secretion of Abeta 1-42 from human as well as rodent neural cells was significantly increased by overexpression of mutant presenilins even under basal culture conditions (1-5). Most importantly, however, overexpression of Par-4 drastically increased the Abeta 1-42/Abeta total ratio in the conditioned media about 6-8 h after trophic factor withdrawal (Fig. 3), indicating that Par-4 may have a significant impact on APP processing under apoptotic conditions. It is noteworthy that the Par-4-induced increase in Abeta 1-42 secretion did not occur until 6-8 h after trophic factor withdrawal (Fig. 3). This is the time when most IMR-32 cells overexpressing Par-4 were still alive and morphologically well preserved, but the apoptotic process should have been irreversibly triggered in most of the cells after trophic factor withdrawal. Time course analysis of caspase activation demonstrated that exacerbated caspase activation induced by Par-4 in IMR-32 cells was apparent as early as 2 h after trophic factor withdrawal (Fig. 5), indicating that Par-4 increases secretion of Abeta 1-42 after initiation of apoptotic cascades. To determine whether the exacerbated caspase activation was responsible for the increase in Abeta 1-42 secretion after trophic factor withdrawal, we examined whether the broad spectrum caspase inhibitor BD-fmk could block the Par-4-induced increase in Abeta 1-42 secreted by IMR-32 cells. Our results clearly demonstrated that the Par-4-induced increase in secretion of Abeta 1-42 is largely a caspase-dependent event.

The effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the dominant negative regulator of Par-4 activity, the leucine zipper domain. The results demonstrate that the actions of Par-4 on APP processing, like its actions in apoptotic pathway (16), require its interaction with other proteins via the leucine zipper domain. The proteins that interact with Par-4 and regulate (directly or indirectly) APP processing are unknown. However, recent study showed that Par-4 interacts with the regulatory domains of the zeta  and lambda  forms of protein kinase C and inhibits their enzyme activity (24). The possible implications of these interactions in APP processing need to be examined carefully because several studies have indicated that protein kinase C-dependent pathways might participate in regulation of APP processing (25-27).

All known genetic factors of familial AD, including mutations in APP and presenilins, lead to increased production of amyloidogenic Abeta 1-42 (1-5). Recent evidence suggests that the presenilin proteins may be gamma -secretase, although their function as gamma -secretase may require help from additional proteins, such as nicastrin (28-32). Interestingly, all three familial Alzheimer's disease genes have also been shown to regulate neuronal apoptosis (1-5), suggesting that dysregulation of apoptotic pathways may play a role in neuronal degeneration in AD. The data provided in this study reconcile the amyloid/apoptosis hypotheses of AD and suggest that abnormal processing of APP and increased production of Abeta 1-42 may occur as a result of initiation and execution of apoptotic cascades, such as aberrant induction of Par-4 expression and activation of caspases after trophic factor withdrawal.

    FOOTNOTES

* This work was supported by grants from the American Federation for Aging Research and the Alzheimer's Association (to Q. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Neurobiology and Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, P. O. Box 95, Rootstown, OH 44272-0095. Tel.: 330-325-6655; Fax: 330-325-5916; E-mail: qguo@neoucom.edu.

Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010996200

    ABBREVIATIONS

The abbreviations used are: Par-4, prostate apoptosis response-4; Leu.zip, leucine zipper domain; Abeta , amyloid beta  peptide; APP, beta -amyloid precursor protein; AD, Alzheimer's disease; ELISA, enzyme linked-immunosorbent assay; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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