Prostate Apoptosis Response-4 Production in Synaptic Compartments Following Apoptotic and Excitotoxic Insults

Evidence for a Pivotal Role in Mitochondrial Dysfunction and Neuronal Degeneration

Authors

  • Wenzhen Duan,

  • Vivek M. Rangnekar,

  • Mark P. Mattson


  • Lippincott Williams & Wilkins, Inc., Philadelphia

  • Abbreviations used: CHX, cycloheximide; DEVD-CHO, biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde; HNE, 4-hydroxynonenal; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Par-4, prostate apoptosis response-4; PBS, phosphate-buffered saline; STS, staurosporine.

Address correspondence and reprint requests to Dr. M. P. Mattson at Sanders-Brown Research Center on Aging, 211 Sanders-Brown Building, 800 South Limestone Street, Lexington, KY 40536, U.S.A.

Abstract

Abstract: Synapses are often located at great distances from the cell body and so must be capable of transducing signals into both local and distant responses. Although progress has been made in understanding biochemical cascades involved in neuronal death during development of the nervous system and in various neurodegenerative disorders, it is not known whether such cascades function locally in synaptic compartments. Prostate apoptosis response-4 (Par-4) is a leucine zipper and death domain-containing protein that plays a role in neuronal apoptosis. We now report that Par-4 levels are rapidly increased in cortical synaptosomes and in dendrites of hippocampal neurons in culture and in vivo, following exposure to apoptotic or excitotoxic insults. Par-4 expression is regulated at the translational level within synaptic compartments. Par-4 antisense treatment suppressed mitochondrial dysfunction and caspase activation in synaptosomes and prevented death of cultured hippocampal neurons following exposure to excitotoxic and apoptotic insults. Local translational regulation of death-related proteins in synaptic compartments may play a role in programmed cell death, adaptive remodeling of synapses, and neurodegenerative disorders.

Apoptosis is a form of cell death characterized by cell shrinkage, maintenance of organellar integrity, and nuclear chromatin condensation and fragmentation (Wyllie et al., 1990). Biochemical features of apoptosis include mitochondrial membrane depolarization (Vayssiere et al., 1994; Kroemer et al., 1997), activation of proteases called caspases (Miller, 1997; Yuan, 1997), and release of factors from mitochondria that induce nuclear DNA fragmentation (Kluck et al., 1997; Kroemer et al., 1997; Yang et al., 1997). In addition to its role in developmental neuron death, apoptosis may occur in several different neurodegenerative conditions, including stroke (Linnik et al., 1993), Alzheimer's disease (Loo et al., 1993; Su et al., 1994; Kruman et al., 1997; Guo et al., 1998), and Huntington's disease (Portera-Cailliau et al., 1995). Many signals between neurons are transduced in synaptic compartments that are often located at great distances from the cell body. Accumulating data suggest that synapses are sites where the cell death process is initiated in various physiological and pathophysiological settings. For example, receptors for neurotrophic factors are concentrated in presynaptic terminals wherein their activation is believed to play a central role in regulation of neuronal survival during development of the nervous system (Oppenheim, 1991), and receptors for the excitatory transmitter glutamate are concentrated in postsynaptic dendritic spines wherein their overactivation may contribute to the neurodegenerative process in various disorders (Choi, 1994; Mattson, 1994; Mattson et al., 1998a, b). Despite such evidence suggesting a critical role for synaptic events in effecting or preventing neuronal death, the nature of local biochemical events that might mediate synaptic degenerative cascades is unknown.

Although apoptosis is believed to be an active form of cell death mediated by production of “killer” proteins, the identification of such proteins has been difficult. We recently identified a 38-kDa protein called prostate apoptosis response-4 (Par-4) whose expression increases in prostate cells undergoing apoptosis (Sells et al., 1997). Analysis of the predicted amino acid sequence of Par-4 reveals both a “death domain” and a leucine zipper domain that likely mediate protein-protein interactions (Diaz-Meco et al., 1996; Sells et al., 1997). Overexpression of full-length Par-4 in PC12 cells increases their vulnerability to apoptosis, whereas overexpression of the Par-4 leucine zipper domain alone exerts a dominant negative action resulting in cellular resistance to apoptosis (Guo et al., 1998). We now report that Par-4 production is induced at the translational level in synaptic compartments following exposure to apoptotic stimuli and that such local up-regulation of Par-4 plays an important role in synaptic mitochondrial dysfunction, caspase activation, and cell death.

MATERIALS AND METHODS

Materials

Staurosporine (STS), 4-hydroxynonenal (HNE), FeSO4, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cycloheximide (CHX), and actinomycin D were obtained from Sigma (St. Louis, MO, U.S.A.). Hoechst 33342 and rhodamine-123 were purchased from Molecular Probes (Eugene, OR, U.S.A.). Par-4 antisense oligodeoxynucleotide (5′-ATAGCCGCCGGTCGCCATGTT-3′) and nonsense oligodeoxynucleotide (5′-CCGTGTCTGATCTTCGTGCGT-3′) were purchased from IDT (Coralville, IA, U.S.A.), and were prepared as 1 mM stocks solution in sterile water.

Synaptosome preparation and hippocampal cell cultures

Female Sprague-Dawley rats (weighing 250-300 g) were killed and decapitated, and brains were removed. Cerebral hemispheres were cut into small fragments and homogenized in a buffer containing 0.32 M sucrose, 4 μg/ml pepstatin, 5 μg/ml aprotinin, 20 μg/ml trypsin inhibitor, 4 μg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, and 20 mM HEPES. Synaptosomes were then prepared using methods described previously (Keller et al., 1997; Mattson et al., 1998a). Previous immunochemical, electron microscopic, and functional analyses have shown that such synaptosome preparations contain no nuclei, are highly enriched in postsynaptic and presynaptic markers, and contain very low levels of astrocytic elements (Keller et al., 1997; Mattson et al., 1998a; Begley et al., 1999). Protein concentrations in synaptosomal preparations were determined (Pierce BCA kit), and equivalent amounts of synaptosomes were aliquoted to wells of 96-well plates or 1.5-ml Eppendorf tubes. All experimental treatments were performed in synaptosomes suspended in Locke's buffer (NaCl, 154 mM; KCl, 5.6 mM; CaCl2, 2.3 mM; MgCl2, 1.0 mM; NaHCO3, 3.6 mM; glucose, 5 mM; and HEPES, 5 mM; pH 7.2). Embryonic rat hippocampal cell cultures were established and maintained as described previously (Mattson et al., 1997). Cells were plated in 35-mm-diameter plastic or glass-bottom dishes on a polyethylenimine substrate in 0.8 ml of medium consisting of minimum essential medium with Earle's salts supplemented with 10% heat-inactivated fetal bovine serum, 1 mM L-glutamine, 1 mM pyruvate, 20 mM KCl, and 26 mM sodium bicarbonate (pH 7.2). Following cell attachment, the culture medium was replaced with Neurobasal medium with B27 supplements (GIBCO). Experiments were performed in 7-9 day-old cultures, an interval during which the neurons express NMDA and AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate receptors and are vulnerable to excitotoxic and metabolic insults (Mattson et al., 1991; Cheng et al., 1995).

Measurements of mitochondrial function

The conversion of the dye MTT to formazan crystals in cells has been shown to be related to mitochondrial redox state (Shearman et al., 1995) and respiratory chain activity (Musser and Oseroff, 1994). MTT was dissolved in phosphate-buffered saline (PBS) at a concentration of 2.5 mg/ml. The MTT solution was mixed with synaptosomes (1:10 vol/vol, MTT/synaptosomes) and incubated for 3 h. The synaptosomes were pelleted by centrifugation at 13,000 rpm for 10 min. The pellet was suspended in solubilization buffer (100% dimethyl sulfoxide), and absorbance (592 nm) of each sample was quantified using a spectrophotometer. The dye rhodamine-123 (Molecular Probes) was used as another measure of mitochondrial function; uptake of rhodamine-123 has been shown to be related to mitochondrial transmembrane potential (Mattson et al., 1993a; Bindokas and Miller, 1995; Kim et al., 1998). In brief, synaptosomes and cultured cells were incubated for 30 min in the presence of 10 μM dye and then were washed twice in Locke's buffer. Following washing, the synaptosomes were seeded into 35-mm-diameter glass-bottom culture dishes and were allowed to settle on the glass surface during a 10-15-min incubation. Fluorescence in synaptosomes and cultured neurons was imaged using a confocal laser scanning microscope with excitation at 488 nm and emission at 510 nm, and the average pixel intensity in user-defined areas corresponding to synaptosomal aggregates or dendrites of cultured hippocampal neurons was determined using Imagespace software (Molecular Dynamics). All images were coded and analyzed without knowledge of experimental treatment history of the synaptosomes or cultured neurons.

Measurement of caspase activation

Caspase activity was assessed in synaptosomes and cultured neurons using methods similar to those described previously (Mattson et al., 1998a), which used the biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO; Calbiochem), a pseudosubstrate and inhibitor of caspases (Gurtu et al., 1997; Margolin et al., 1997). At designated time points following experimental treatment, synaptosomes or cultured cells were incubated for 10 min in Locke's buffer containing 0.01% digitonin. Synaptosomes and cells were then incubated for 20 min in the presence of 10 μg/ml DEVD-CHO, washed three times with PBS (2 ml per wash), and fixed for 30 min in a cold solution of 4% paraformaldehyde in PBS. Synaptosomes and cells were then incubated for 5 min in PBS containing 0.2% Triton X-100, followed by a 30-min incubation in PBS containing 5 μg/ml Oregon Green-streptavidin (Molecular Probes). Synaptosomes and cells were then washed twice with PBS, and images of fluorescence (corresponding to conjugates of activated caspase with DEVD-CHO) were acquired using a confocal laser scanning microscope. Levels of fluorescence in synaptosomal clusters or in neuronal cell bodies and neurites were quantified using Imagespace software (Molecular Dynamics) as described previously (Mattson et al., 1998a).

Quantification of neuron survival and apoptosis

These methods are detailed in previous studies (Mattson et al., 1991; Kruman et al., 1997). In brief, viable neurons were counted in premarked microscope fields (10× objective) before experimental treatment and different time points after treatment. Viability of the remaining neurons was assessed by morphological criteria. Neurons with intact neurites of uniform diameter and a soma with a smooth appearance were considered viable, whereas neurons with damaged neurites and a shrunken soma were considered nonviable. To assess apoptosis, cells were fixed in 4% paraformaldehyde and stained with the DNA-binding dye Hoechst 33342. Cells were visualized under epifluorescence illumination (340 nm excitation and 510 nm barrier filter) with a 40× oil immersion objective, and the percentage of cells with condensed and fragmented DNA was determined (cells in five to 10 random fields/cultures were scored, and counts were made in at least four separate cultures per treatment condition).

Immunocytochemistry and western blot analyses

The methods were similar to those described previously (Mattson et al., 1997; Guo et al., 1998). In brief, cells were fixed in 4% paraformaldehyde, membranes were permeabilized by exposure for 5 min to 0.2% Triton X-100 in PBS, and cells were placed in blocking serum (5% goat serum in PBS) for 30 min. Cells were then exposed to primary antibody (anti-Par-4 rabbit polyclonal antibody, 1:4,000) (Guo et al., 1998) overnight at 4°C, followed by an incubation for 1 h with biotinylated goat anti-rabbit secondary antibody (1:200) and 30 min in the presence of fluorescein isothiocyanate-avidin (Vector Labs, Burlingame, CA, U.S.A.). Images of Par-4 immunofluorescence were acquired using a confocal laser scanning microscope with a 60× oil immersion objective (488 nm excitation and 510 nm emission). All images were acquired using the same laser intensity and photodetector gain to allow quantitative comparisons of relative levels of immunoreactivity in neurons and dendrites; the average pixel intensity within regions of interest was determined using the Imagespace software provided by the manufacturer (Molecular Dynamics). Determinations were made in four separate cultures. All images were analyzed without knowledge of treatment history. For double labeling, cells were incubated in PBS containing anti-synaptophysin mouse monoclonal antibody (1:250; StressGen) and anti-Par-4 rabbit polyclonal antibody overnight at 4°C. Cells were then washed with PBS and incubated for 30 min in PBS containing biotinylated horse anti-mouse secondary antibody (1:200), followed by a 30-min incubation in PBS containing Texas Red-labeled goat anti-rabbit secondary antibody (1:200; Molecular Probes). Cells were then washed with PBS, incubated for 30 min in the presence of fluorescein isothiocyanate-avidin, and washed with water. Images were acquired using confocal laser scanning microscope with a 60× oil immersion objective.

For western blot analysis, 50 μg of solubilized proteins was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel) and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C in the presence of 5% nonfat milk and then incubated for 2 h with Par-4 antibody (1:1,000). The membrane was then exposed for 1 h to horseradish peroxidase-conjugated secondary antibody (1:3,000; Jackson ImmunoResearch Labs, West Grove, PA, U.S.A.) and immunoreactive protein was visualized using a chemiluminescence-based detection kit according to the manufacturer's protocol (ECL kit; Amersham Corp., Arlington Heights, IL, U.S.A.).

Kainic acid lesions, tissue processing, and immunohistochemistry

Adult male Sprague-Dawley rats (weighing, 220-240 g) were maintained under temperature- and light-controlled conditions (20-23°C and a 12-h light:12-h dark cycle with lights on at 7:00 and off at 19:00); they had free access to food and water. Kainic acid (Sigma) was dissolved in saline and injected intraperitoneally (10 mg/kg); the control group was injected with the same volume of saline. All procedures were in strict accordance with the NIH Guidelines for Animal Care and Use and were approved by the University of Kentucky Animal Care and Use Committee. At designated time points following kainic acid administration, the rats were anesthetized with halothane and then either decapitated (for preparation of synaptosomes) or were perfused through the ascending aorta with physiological saline followed by 4% paraformaldehyde in PBS (pH 7.4) (for histological analyses). Fixed brains were cryoprotected in a 30% sucrose solution, and coronal brain sections (30 μm) were cut on a freezing microtome. Sections were either stained with cresyl violet or were immunostained with Par-4 antibody (1:4,000) using methods described previously (Smith-Swintosky et al., 1996; Bruce-Keller et al., 1998).

RESULTS

Exposure of synaptosomes to oxidative and apoptotic insults results in rapid, translation-dependent increases of Par-4 levels and mitochondrial dysfunction

Western blot analysis showed that Par-4 is present in cortical synaptosomes (Fig. 1). Exposure of synaptosomes to Fe2+ (an inducer of membrane lipid peroxidation) (Goodman et al., 1996), HNE (a neurotoxic aldehydic product of lipid peroxidation) (Kruman et al., 1997; Mark et al., 1997), or STS (a bacterial alkaloid that induces neuronal apoptosis) (Koh et al., 1995; Kruman et al., 1998) resulted in rapid and progressive increases in levels of Par-4 (Fig. 1). With each insult, Par-4 levels increased within 2 h of exposure and continued to increase through 6 h of exposure. Because synaptosomes lack nuclei, the increase in Par-4 protein levels could not be due to increased gene transcription. We therefore sought to determine whether the insult-induced increases of Par-4 levels were the result of increased translation of Par-4 in the synaptosomes. Treatment of synaptosomes with the protein synthesis inhibitor CHX (10 μg/ml) largely prevented the increase in Par-4 protein levels otherwise seen following exposure to Fe2+, HNE, and STS (Fig. 2A). In contrast, actinomycin D, an inhibitor of transcription, had no effect on basal or insult-induced Par-4 levels in synaptosomes (data not shown).

Figure 1.

Par-4 is present is synaptosomes wherein its levels are increased following exposure to oxidative and apoptotic insults. Synaptosomes were exposed to 0.2% dimethyl sulfoxide (Con) for 4 h or were exposed for the indicated intervals to 10 μM FeSO4 (Fe), 10 μM HNE, or 100 nM STS. Western blots were then performed on synaptosomal homogenates (50 μg of protein per lane). Similar results were obtained in two additional experiments.

Figure 2.

CHX blocks the increase in Par-4 levels and preserves mitochondrial function in synaptosomes exposed to oxidative and apoptotic insults. A: Synaptosomes were pretreated for 2 h with saline (Control) or 10 μg/ml CHX. Synaptosomes were then exposed for 6 h to 10 μM FeSO4 (Fe), 10 μM HNE, or 100 nM STS. Western blots were then performed on synaptosomal homogenates (50 μg of protein per lane). Similar results were obtained in two additional experiments. B: Synaptosomes were pretreated for 2 h with saline (Control) or 10 μg/ml CHX. Synaptosomes were then exposed for 6 h to vehicle (0.2% dimethyl sulfoxide), 10 μM Fe2+, 100 nM STS, or 10 μM HNE, and levels of MTT reduction were quantified. Data are mean ± SD (bars) values of determinations made in four synaptosome preparations. *p < 0.01 compared with control (Vehicle) value; **p < 0.05 compared with value for Fe2+, HNE, or STS alone, respectively. C: Synaptosomes were pretreated for 2 h with saline (Control) or 10 μg/ml CHX. Synaptosomes were then exposed for 6 h to 10 μM Fe2+, 10 μM HNE, or 100 nM STS, and levels of rhodamine-123 fluorescence were quantified. Data are mean ± SD (bars) values of determinations made in four separate synaptosome preparations. *p < 0.01 compared with control (Vehicle) value; **p < 0.01 compared with value for Fe2+, HNE, or STS alone, respectively, by ANOVA with Scheffé's post hoc tests.

FIG. 1.

FIG. 2.

Previous studies have shown that impaired mitochondrial function occurs following exposure of synaptosomes to oxidative insults, including Fe2+ and HNE (Keller et al., 1997). In intact cells undergoing apoptosis, mitochondrial membrane depolarization and decreased respiratory chain activity may be required for subsequent nuclear disintegration and cell death (Richter, 1993; Werth and Thayer, 1994; Kroemer et al., 1997). We therefore measured two different parameters of mitochondrial function: MTT reduction (a measure redox state and energy charge) and rhodamine-123 fluorescence (a measure of mitochondrial transmembrane potential) in synaptosomes. Highly significant 50-60% decreases in levels of MTT reduction (Fig. 2B) and rhodamine-123 fluorescence (Fig. 2C) were evident within 6 h of exposure of synaptosomes to Fe2+ (10 μM), HNE (10 μM), and STS (100 nM). Pretreatment of synaptosomes with CHX significantly attenuated the decreases in MTT reduction and rhodamine-123 fluorescence otherwise induced by Fe2+, HNE, and STS (Fig. 2B and C), demonstrating a requirement for protein synthesis in the mitochondrial dysfunction.

Par-4 antisense oligonucleotide treatment attenuates mitochondrial dysfunction and caspase activation in synaptosomes

Because Par-4 levels were increased relatively rapidly following exposure of synaptosomes to the different insults and because a recent study suggested a role for Par-4 in mitochondrial dysfunction in PC12 cells undergoing apoptosis (Guo et al., 1998), we examined the possibility that early induction of Par-4 is causally linked to synaptic mitochondrial dysfunction. To this end, we used a Par-4 antisense oligodeoxynucleotide that was previously shown to suppress Par-4 expression in hippocampal neurons exposed to apoptotic insults (Guo et al., 1998). Western blot analysis showed that the Par-4 antisense oligonucleotide had no detectable effect on levels of Par-4 protein during a 6-h exposure period under basal conditions (Fig. 3A). In contrast, Par-4 antisense treatment completely blocked the increases in Par-4 protein levels otherwise induced by Fe2+, HNE, and STS. A control nonsense oligonucleotide did not prevent Par-4 induction by any of the insults, demonstrating specificity of the effect of Par-4 antisense oligonucleotide (Fig. 3A). We next determined the effects of Par-4 antisense on levels of MTT reduction and rhodamine-123 fluorescence in synaptosomes exposed to oxidative and apoptotic insults. Fe2+, HNE, and STS each caused significant decreases in levels of MTT reduction and rhodamine-123 fluorescence during 6-h exposures in control synaptosomes pretreated with either vehicle or nonsense oligonucleotide; in contrast, mitochondrial functional parameters were largely preserved following exposure to these insults in synaptosomes pretreated with Par-4 antisense oligonucleotide (Fig. 3B and C). Collectively, these data suggest a critical role for increased Par-4 synthesis in mitochondrial dysfunction that occurs in synaptic compartments during oxidative and apoptotic insults.

Figure 3.

Suppression of Par-4 induction by a Par-4 antisense oligonucleotide (AS) preserves mitochondrial function following exposure of synaptosomes to oxidative and apoptotic insults. A: Western blot analyses of Par-4 protein levels in synaptosomes (50 μg of protein per lane for all blots). Upper blot: Synaptosomes were left untreated (Control), were treated for 6 h with nonsense oligonucleotide (NS), or were treated with 25 μM Par-4 AS for 1, 2, 3, 4, 5, or 6 h. Middle and lower blots: Synaptosomes were pretreated for 2 h with 25 μM NS or 25 μM Par-4 AS. Synaptosomes were then exposed for 4 h (middle blot) or 6 h (lower blot) to 0.2% dimethyl sulfoxide (Control), 10 μM FeSO4 (Fe), 100 nM STS, or 10 μM HNE. B and C: Synaptosomes were pretreated for 2 h with saline (Control), 25 μM NS, or 25 μM Par-4 AS. Synaptosomes were then exposed for 6 h to vehicle, 10 μM Fe2+, 10 mM HNE, or 100 nM STS, and levels of (B) MTT reduction or (C) rhodamine-123 fluorescence were quantified. Data are mean ± SD (bars) values of determinations made in four synaptosome preparations. *p < 0.01 compared with the corresponding control (Vehicle) values; **p < 0.05 compared with the corresponding Control and NS values by ANOVA with Scheffé's post hoc tests.

FIG. 3.

Caspases are proteases that play pivotal roles in the effector phase of apoptosis. We recently reported that caspase-3 is present in rat cortical synaptosomes, wherein it is activated following exposure to staurosporine and amyloid β-peptide (Mattson et al., 1998a, b). Because Par-4 levels increased before mitochondrial dysfunction following exposure of synaptosomes to oxidative and apoptotic insults and because mitochondrial alterations may trigger caspase activation in neurons undergoing apoptosis (Armstrong et al., 1997), we determined whether the increase in Par-4 levels was required for caspase activation. Exposure of synaptosomes to Fe2+, HNE, or STS for 6 h resulted in significant increases in caspase activity as indicated by an increase in DEVD-associated fluorescence (Fig. 4). Pretreatment of synaptosomes with Par-4 antisense oligonucleotide completely prevented the increase in caspase activity caused by each insult, whereas nonsense DNA was ineffective. These data suggest that Par-4 induction is an early event in the apoptotic cascade engaged at synapses that plays an active role in promoting mitochondrial dysfunction and caspase activation.

Figure 4.

Par-4 antisense oligonucleotide (AS) treatment inhibits oxidative stress- and STS-induced caspase activation in synaptosomes. Synaptosomes were pretreated for 2 h with 25 μM nonsense oligonucleotide (NS) or 25 μM Par-4 AS. Synaptosomes were then exposed for 4 h to 0.2% dimethyl sulfoxide (Control), 10 μM FeSO4 (Fe2+), 10 μM HNE, or 100 nM STS, and relative levels of DEVD-associated fluorescence (a measure of caspase activation) were quantified. Data are mean ± SD (bars) values of determinations made in four separate synaptosome preparations. *p < 0.01 compared with the corresponding control value; **p < 0.01 compared with the corresponding NS values by ANOVA with Scheffé's post hoc tests.

FIG. 4.

Par-4 is induced in neurites and synaptic regions of cultured hippocampal neurons following exposure to apoptotic and excitotoxic insults and following kainate-induced seizures in vivo

To determine whether Par-4 is induced in synaptic compartments of intact neuronal circuits, we used mature primary hippocampal cell cultures in which neurons form extensive glutamatergic synapses (Mattson et al., 1991, 1998a). Neurons in such cultures are vulnerable to excitotoxicity (Mattson et al., 1991) and to apoptosis induced by STS (Koh et al., 1995; Prehn et al., 1997). Within 4 h of exposure of hippocampal cultures to glutamate (10 μM) and STS (100 nM), levels of Par-4 immunoreactivity were greatly increased in neurites and cell bodies of neurons (Fig. 5). To determine whether Par-4 levels were increased in synaptic compartments, we performed confocal analysis of cells double-labeled with antibodies against Par-4 and the synaptic vesicle-associated protein synaptophysin (Horner et al., 1996). Analyses were performed in vehicle-treated control cultures and in cultures exposed to glutamate or STS. Examination of the confocal images of cells double-stained with Par-4 and synaptophysin antibodies revealed colocalization of Par-4 immunoreactivity and synaptophysin immunoreactivity in punctata along the length of neurites (Fig. 5A). Consistent with previous studies (Mundigl et al., 1993), we observed considerable synaptophysin immunoreactivity in the cell body. Based on our previous studies of prostate cells (Sells et al., 1997) and PC12 cells (Guo et al., 1998) and the images of primary hippocampal neurons (Fig. 5A), it appears that Par-4 is predominantly a cytoplasmic protein and that its distribution is not markedly altered in cells undergoing apoptosis. The images indicate that Par-4 was present and that its levels were increased following exposure to excitotoxic and apoptotic insults, in synaptic compartments as well as in the cell body.?

Figure 5a.

Excitotoxic and apoptotic insults induce increased Par-4 levels in dendrites and synaptic regions of cultured hippocampal neurons. Hippocampal cultures were exposed for 4 h to 0.2% dimethyl sulfoxide (Control), 10 μM glutamate, or 100 nM STS. Upper panels: Cells were then fixed and immunostained with Par-4 antibody alone. Lower panels: Cells were double-stained using Par-4 and synaptophysin antibodies; the confocal images are anaglyphs showing Par-4 immunoreactivity (red), synaptophysin immunoreactivity (green), and regions of colocalization of Par-4 and synaptophysin immunoreactivities (yellow).

Figure 5b.

Excitotoxic and apoptotic insults induce increased Par-4 levels in dendrites and synaptic regions of cultured hippocampal neurons. Hippocampal cultures were exposed to 0.2% dimethyl sulfoxide (control), 10 μM glutamate, or 100 nM STS. Cells were then fixed and immunostained with Par-4 antibody, and confocal images of fluorescence were acquired. Fluorescence intensity was quantified in regions of interest (see Materials and Methods), and data are mean ± SD (bars) values of determinations made in four separate cultures (15-25 neurons analyzed per culture). *p < 0.01 compared with the corresponding control value by ANOVA with Scheffé's post hoc tests.

FIG. 5A.

FIG. 5B.

To determine whether Par-4 is also induced by excitotoxic stimuli in vivo, we administered the seizure-inducing excitotoxin kainate to adult rats. Immunohistochemical analyses showed that under basal conditions Par-4 immunoreactivity was weak or absent in the hippocampus (Fig. 6A). Par-4 immunoreactivity was increased in molecular layers of regions CA1 and CA3 of the hippocampus and to a lesser extent in the molecular layers of the dentate gyrus, at 24 and 72 h following kainate administration (Fig. 6A). Examination of adjacent sections stained with cresyl violet revealed little or no neuronal loss 24 h following kainate and extensive loss of CA3 neurons 72 h postkainate. In an additional experiment, rats were administered either saline or kainate and killed 24 or 72 h later. Hippocampi were then removed, and synaptosomes were prepared. Synaptosomal proteins were subjected to western blot analysis using the Par-4 antibody. Levels of Par-4 protein were greatly increased in synaptosomes 24 and 72 h following kainate administration (Fig. 6B).

Figure 6.

Par-4 levels are increased in hippocampal cells following kainate (KA)-induced seizures in adult rats. Rats were administered saline and killed 24 h later (control) or were administered 10 mg/kg KA and killed 24 or 72 h later. Either brains were perfused for histological analyses, or hippocampi were rapidly removed and prepared for western blot analysis. A: Coronal sections of hippocampus were stained with cresyl violet (left) or were immunostained with Par-4 antibody (right). Note the loss of CA3 neurons 72 h following KA administration and increased Par-4 immunoreactivity within 24 h of KA administration. B: Western blot analysis shows increased Par-4 levels in synaptosomes at 24 and 72 h post-KA. Con, control.

FIG. 6.

Par-4 antisense oligonucleotide protects cultured hippocampal neurons against neuritic degeneration and cell death induced by excitotoxic and apoptotic insults: attenuation of mitochondrial dysfunction and caspase activation

To determine whether Par-4 plays a role in excitotoxic and apoptotic neuronal death, we pretreated cultures with Par-4 antisense or nonsense DNA, exposed them to glutamate or STS, and then quantified neuron survival at 12, 24, and 48 h. A highly significant attenuation of glutamate- and STS-induced neuronal death was seen in cultures pretreated with Par-4 antisense DNA compared with cultures treated with control nonsense DNA (Fig. 7A). In an additional experiment, cultures were exposed to glutamate or STS for 24 h and were then fixed and stained with the fluorescent DNA-binding dye Hoechst 33342. Counts of neurons with apoptotic nuclei showed that Par-4 antisense oligonucleotide treatment almost completely prevented apoptosis induced by glutamate and STS (Fig. 7B). We next pretreated cultures with either Par-4 antisense or nonsense DNA, exposed the cultures to glutamate or STS for 8 h, and then measured levels of rhodamine-123 fluorescence in neurites of the hippocampal neurons. Glutamate and STS each caused a highly significant decrease in levels of neuritic rhodamine-123 fluorescence in control cultures, and the decrease in rhodamine-123 fluorescence was almost completely prevented in neurons pretreated with Par-4 antisense oligonucleotide (Fig. 7C and D).

Figure 7.

Par-4 antisense oligonucleotide (AS) treatment protects cultured hippocampal neurons against excitotoxicity and apoptosis and stabilizes mitochondrial function. A: Cultures were pretreated for 2 h with Par-4 AS or nonsense oligonucleotide (NS) and then were exposed to 0.2% dimethyl sulfoxide (Control), 10 μM glutamate (Glu), or 100 nM STS. Neuronal survival was quantified at the indicated time points following exposure. Data are mean ± SD (bars) values of determinations made in four separate cultures. B: Cultures were pretreated for 2 h with Par-4 AS or NS and were then exposed for 24 h to 0.2% dimethyl sulfoxide (Control), 10 μM Glu, or 100 nM STS. Cells were fixed and stained with the DNA-binding dye Hoechst 33342, and the percentages of neurons in each culture exhibiting apoptotic nuclei was determined. Data are mean ± SD (bars) values of determinations made in four separate cultures. *p < 0.01 compared with the corresponding control values; **p < 0.01 compared with the corresponding NS values. C: Cultures were pretreated for 2 h with Par-4 AS or NS and then were exposed for 24 h to 0.2% dimethyl sulfoxide (control), 10 μM Glu, or 100 nM STS. Cells were then loaded with rhodamine-123, and levels of fluorescence in dendrites were quantified (see Materials and Methods). Data are mean ± SD (bars) values of determinations made in four separate cultures. *p < 0.01 compared with the corresponding control values; **p < 0.01 compared with the corresponding NS values by ANOVA with Scheffé's post hoc tests. D: Confocal laser scanning microscope images of rhodamine-123 fluorescence in hippocampal neurons. Cultures were pretreated for 2 h with Par-4 AS or NS and then were exposed for 8 h to 0.2% dimethyl sulfoxide (control), 10 μM Glu, or 100 nM STS. Note that Glu and STS caused decreases in rhodamine-123 fluorescence in dendrites and cell bodies of neurons in NS-treated control cultures but not in neurons pretreated with Par-4 AS.

FIG. 7.

Because Par-4 antisense oligonucleotide treatment suppressed STS-induced caspase activation in synaptosomes, we determined whether Par-4 also played a role in caspase activation in intact neurons. Hippocampal cultures were pretreated with Par-4 antisense or nonsense oligonucleotide and were then exposed to glutamate or STS for 8 h. Cells were then processed for in situ localization of activated caspase. Confocal images showed that both glutamate and STS caused large increases in levels of caspase activation in neurites and cell bodies of neurons in control cultures pretreated with nonsense oligonucleotide (Fig. 8). In contrast, very little or no increase in levels of activated caspase occurred in neurites or cell bodies of neurons in cultures pretreated with Par-4 antisense oligonucleotide (Fig. 8), suggesting a pivotal role for Par-4 induction in the cascade of events leading to caspase activation in response to excitotoxic and apoptotic insults.

Figure 8.

Hippocampal cultures were pretreated for 2 h with Par-4 antisense (AS) or nonsense (NS) oligonucleotide and were then exposed for 8 h to 0.2% dimethyl sulfoxide (control), 10 μM Glu, or 100 nM STS. Cells were then processed for localization of activated caspases. Note that both STS and glutamate caused pronounced increases in caspase activation along the length of dendrites, as well as in the cell body. Par-4 AS treatment largely prevented the increase of DEVD fluorescence in dendrites and cell body.

FIG. 8.

DISCUSSION

Our data provide new insight into the compartmentalization of biochemical cascades that underlie degeneration of neuronal circuits and suggest that such cascades may be involved in excitotoxic and apoptotic neuronal death. The demonstration of translation-mediated expression of Par-4 in synaptosomes following exposure to oxidative and apoptotic insults provides direct evidence for local induction of a cell death-associated protein in synaptic compartments. Previous studies have shown that the translational machinery is present in rat cortical synaptosomes (Gilbert, 1972; Rao and Steward, 1991), in dendrites and synaptic spines of cultured hippocampal neurons (Steward, 1995; Tiedge and Brosius, 1996), and in isolated growth cones (Davis et al., 1992). However, local production of a specific protein linked to cell death in synaptic compartments has not previously been reported. The ability of Par-4 antisense oligonucleotide treatment to suppress mitochondrial dysfunction and caspase activation in synaptosomes following exposure to oxidative insults and STS and in dendritic compartments of cultured hippocampal neurons following exposure to glutamate suggests a pivotal role for Par-4 at a quite early step in the cascade of events that leads to synaptic/dendritic degeneration and cell death. Presumably, Par-4 antisense DNA binds to Par-4 mRNA in synaptic compartments and dendrites and prevents translation of the Par-4 mRNA. Indeed, our western blot analysis showed that Par-4 antisense oligonucleotide (but not a nonsense oligonucleotide) completely blocked the increase in Par-4 protein levels following exposure of synaptosomes to oxidative insults and STS. In addition, the fact that Par-4 antisense oligonucleotide treatment had no detectable effect on the basal level of Par-4 during a 4-h exposure period suggests that the half-life of Par-4 is relatively long.

The specific mechanism whereby Par-4 promotes mitochondrial dysfunction, caspase activation, and neuronal death is unknown. However, available data suggest possible roles for one or more of the putative functional domains of Par-4, including the leucine zipper domain and the death domain. Proteins containing leucine zipper domains usually participate in protein-protein interactions, and immunoprecipitation analyses suggest that this is true of Par-4 (Johnstone et al., 1996). A role for the leucine zipper domain in the apoptotic action of Par-4 is suggested by studies of prostate tumor cells (Sells et al., 1997) and PC12 cells (Guo et al., 1998) in which overexpression of a deletion mutant of Par-4 lacking the leucine zipper domain does not enhance apoptosis, in contrast to overexpression of full-length Par-4. Moreover, overexpression of just the leucine zipper domain of Par-4 acts in a dominant negative manner to prevent apoptosis (Sells et al., 1997; Guo et al., 1998). Two proteins that interact with Par-4 are atypical forms of protein kinase C (Diaz-Meco et al., 1996) and the Wilms tumor suppressor protein (Johnstone et al., 1996). Activation of protein kinase Cζ has been reported to prevent apoptosis in tumor cell lines (Berra et al., 1997; Murray and Fields, 1997; Puls et al., 1997), and Par-4 may inhibit protein kinase Cζ (Diaz-Meco et al., 1996). It is not known whether protein kinase Cζ and/or the Wilms tumor suppressor protein play a role in the apoptotic action of Par-4 in neurons. Death domains are contained in the cytoplasmic regions of cell surface receptors that transduce apoptotic signals from cytokines such as tumor necrosis factor and Fas ligand (Schulze-Osthoff et al., 1998). As with other death domain-containing proteins, it will be of considerable interest to elucidate the specific mechanism whereby these proteins engage cascades leading to mitochondrial dysfunction and apoptosis.

Our data showing that glutamate induces increased Par-4 levels in dendrites and synaptic compartments of mature neuronal circuits in hippocampal cultures and that Par-4 levels increase in dendritic fields of hippocampal neurons following kainate-induced seizures in adult rats demonstrate that vigorous activation of glutamate receptors is a potent stimulus for Par-4 induction. These findings suggest that increases in Par-4 levels may occur predominantly in postsynaptic dendritic compartments, although they do not exclude increases in presynaptic terminals. It is interesting that in addition to increasing in molecular layers of the vulnerable CA3 and CA1 regions of hippocampus following kainate administration, Par-4 levels also increased (albeit to a lesser extent) in the molecular layers of the dentate gyrus. The latter observation indicates that seizure activity can induce Par-4 production in neurons, without those neurons dying; this may suggest that different neurons exhibit different thresholds for Par-4-mediated cell death. The ability of Par-4 antisense oligonucleotide treatment to protect against glutamate-induced mitochondrial dysfunction in cultured hippocampal neurons suggests a critical role for Par-4 in mediating excitotoxic neurodegenerative cascades. Although the actions of Par-4 that promote apoptosis remain to be defined, two likely consequences of increased Par-4 expression are perturbed cellular calcium homeostasis and increased levels of oxidative stress. We previously showed that Par-4 plays a central role in calcium ionophore-induced apoptosis of prostate cells (Sells et al., 1997). Calcium influx through NMDA receptors and voltage-dependent channels (Choi, 1994) and generation of reactive oxygen species (Lafon-Cazal et al., 1993; Mattson et al., 1995) are central to the excitotoxic mechanism. Moreover, oxidative stress and perturbed calcium homeostasis play pivotal roles in the neuronal death process induced by each of the other insults used in the present study, Fe2+, HNE, and STS (Koh et al., 1995; Goodman et al., 1996; Kruman et al., 1997, 1998). Par-4 may therefore contribute to neuronal apoptosis by enhancing oxidative stress, caspase activation, and mitochondrial dysfunction.

Finally, the present findings may provide new insight into the pathogenesis of neurodegenerative disorders that involve excitotoxic and apoptotic neuronal death. Excitotoxicity and apoptosis have each been implicated in both acute neurodegenerative conditions such as stroke (Linnik et al., 1993; Choi, 1994), traumatic brain injury (Conti et al., 1998), and severe epileptic seizures (Choi, 1994) and in chronic neurodegenerative disorders including Alzheimer's disease (Loo et al., 1993; Mattson et al., 1993b; Guo et al., 1998), Parkinson's disease (Burke, 1998), Huntington's disease (Portera-Cailliau et al., 1995), and amyotrophic lateral sclerosis (Troost et al., 1995). Our data show that Par-4 is induced in synaptic and dendritic compartments following exposure of neurons to excitotoxic and oxidative insults relevant to several of these disorders and suggest that Par-4 serves as a critical link in the chain of events leading to synaptic dysfunction and degeneration. Although a causal role for Par-4 in human neurodegenerative disorders remains to be established, it was recently reported that Par-4 levels are greatly increased in brain tissue from Alzheimer's patients in association with degenerating neurons (Guo et al., 1998). The latter study also showed that amyloid β-peptide, a protein linked to the neurodegenerative process in Alzheimer's disease, can induce Par-4 expression in cultured neurons. A better understanding of local, transcription-independent regulation of apoptotic cascades in synaptic compartments is likely to reveal novel therapeutic targets for many different neurodegenerative disorders.

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