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Keywords:

  • amyloid β-peptide 31–35 (Aβ[31–35]);
  • apoptosis;
  • cortical neurons;
  • group III metabotropic glutamate receptors (mGluRs);
  • neuroprotection;
  • caspase-dependent intrinsic and extrinsic pathways;
  • PKA-dependent pathway

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

This study aims to investigate the roles of the protein kinase A (PKA)- and caspase-dependent pathways in amyloid β-peptide 31–35 (Aβ[31–35])-induced apoptosis, and the mechanisms of neuroprotection by group III metabotropic glutamate receptor (mGluR) activation against apoptosis induced by Aβ[31–35] in cortical neurons. We demonstrated that Aβ[31–35] induces neuronal apoptosis as well as a significant increase in caspase-3, -8 and -9. Activation of group III mGluRs by l-serine-O-phosphate and (R,S)-4-phosphonophenylglycine (two group III mGluR agonists), which attenuate the effects of Aβ[31–35], provides neuroprotection to the cortical neurons subjected to Aβ[31–35]. We also showed that Rp-cAMP, an inhibitor of cAMP-dependent PKA, has the ability to protect neurons from Aβ[31–35]-induced apoptosis and to reverse almost completely the effects of Aβ[31–35] on the activities of caspase-3. Further, we found that Sp-cAMP, an activator of cAMP-dependent PKA, can significantly abolish the l-serine-O-phosphate- and (R,S)-4-phosphonophenylglycine-induced neuroprotection against apoptosis, and decrease caspase-3, -8 and -9 in the Aβ[31–35]-treated neurons. Our findings suggest that neuronal apoptosis induced by Aβ[31–35] is mediated by the PKA-dependent pathway as well as the caspase-dependent intrinsic and extrinsic apoptotic pathways. Activation of group III mGluRs protects neurons from Aβ[31–35]-induced apoptosis by blocking the caspase-dependent pathways. Inhibition of the PKA-dependent pathway might also protect neurons from Aβ[31–35]-induced apoptosis by blocking the caspase-dependent pathways. Taken together, our observations suggest that Aβ[31–35] might have the ability to activate PKA, which in turn activates the caspase-dependent intrinsic and extrinsic apoptotic pathways, inducing apoptosis in the cortical neurons.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Apoptosis has been implicated in the pathogenesis of various neurodegenerative disorders including AD (Gupta et al., 2006). Alzheimer's disease is characterized by the presence of senile plaques in some brain regions, with a high concentration particularly in those zones where neurodegeneration occurs (Selkoe, 1994). The major protein component of the plaques is the amyloid β-peptide (AβP). This peptide is known to cause the activation of apoptotic cascades, leading to neuronal death in AD (Hardy & Higgins, 1992; Haass & Selkoe, 1993; Lynch et al., 2000; Vickers et al., 2000; Wellington & Hayden, 2000; Awasthi et al., 2005; Heredia et al., 2006; Yao et al., 2007).

Studies have demonstrated that Aβ[1–42] (the first 42 amino acid residues of AβP) and Aβ[25–35] may induce caspase-dependent apoptosis. Apoptotic cell death associated with the activation of caspases has been found in several neuronal cell types exposed to AβP (Mattson et al., 1998; Allen et al., 1999, 2001; Troy et al., 2000; Casley et al., 2002; Wei et al., 2002; Lu et al., 2003; Movsesyan et al., 2004; Matsui et al., 2006). Studies have also shown that activation of the mGluR has the ability to inhibit caspase-dependent apoptosis induced by Aβ[25–35] or Aβ[1–42] in primary neuronal cultures (Troy et al., 2000; Lea & Faden, 2003; Movsesyan et al., 2004). Our recent studies confirmed that Aβ[31–35], which is a shorter sequence of AβP, can also induce apoptosis in the cortical and hippocampal neurons as Aβ[25–35] does (Yan et al., 1999, 2000; Qi et al., 2004), and that group III mGluR agonists, L-SOP and (R,S)-PPG, can protect the cortical neurons from Aβ[31–35]-induced apoptosis. Apoptosis induced by Aβ[31–35] has also been demonstrated in neuronal PC 12 cells (Clementi & Misiti, 2005; Misiti et al., 2005) and rat cerebellar granule cells (Misiti et al., 2006). However, it is unknown whether the neuroprotection of the group III mGluR activation against cortical neurons from Aβ[31–35]-induced apoptosis is associated with the activities of caspases.

In addition, it has been demonstrated that there is a significant increase of cAMP levels in the cerebrospinal fluid from patients of AD, suggesting that the up-regulation of the cAMP-signaling pathway may be implicated in the physiopathology of AD (Martinez et al., 1999). An in vitro study supported this possibility by showing that treatment of hippocampal neurons with Aβ[25–35] can induce an increase in cAMP levels (Prapong et al., 2001). The involvement of the cAMP–protein kinase A (PKA) pathway has also been reported in Aβ[1–42] toxicity in primary neuronal cultures (Echeverria et al., 2005), allicin-induced cell death (Park et al., 2005) and anisomycin-induced apoptosis (Chiarini et al., 2002). These facts suggest a role of the cAMP–PKA pathway in AβP-induced apoptosis. However, it is unknown whether the cAMP–PKA pathway is associated with the Aβ[31–35]-induced apoptosis.

In the present study, we therefore examined the effects of Aβ[31–35] on the activities of caspase-3, -8 and -9 in cortical neurons to determine whether the neuronal apoptosis induced by Aβ[31–35] is associated with changes in the activities of caspases, and if so, whether these changes can be blocked by the activation of group III mGluRs by using L-SOP and (R,S)-PPG, two group III mGluR agonists. We also examined the effects of Sp-cAMP, a potent membrane-permeable activator of cAMP-dependent PKA, and Rp-cAMP, a specific membrane-permeable inhibitor of cAMP-dependent PKA, on Aβ[31–35]-induced apoptosis and changes in the activities of caspase-3, -8 and -9 to find out whether the Aβ[31–35]-induced apoptosis is associated with the activation of the cAMP–PKA pathway. Because a potential link between cAMP–PKA and glutamate had been reported (Figiel et al., 2003), we also investigated the effects of Sp-cAMP on the L-SOP- and (R,S)-PPG-induced changes in apoptosis and activities of caspase-3, -8 and -9, and the effects of Rp-cAMP on Annexin–FITC fluorescence intensity and caspase-3 in Aβ[31–35]-treated cortical neurons to determine whether there is any connection between the PKA-dependent and caspase-dependent pathways.

Our findings demonstrated that neuronal apoptosis induced by Aβ[31–35] is mediated by the caspase-dependent intrinsic and extrinsic apoptotic pathways as well as the PKA-dependent pathway. Activation of group III mGluRs protects neurons from Aβ[31–35]-induced apoptosis by blocking the caspase-dependent pathways. Also, cortical neurons could be protected from Aβ[31–35]-induced apoptosis by the inhibition of the PKA-dependent pathway. In addition, our observations suggested that Aβ[31–35] might have the ability to activate the PKA first, which in turn activates the caspase-dependent intrinsic and extrinsic apoptotic pathways, inducing apoptosis in the cortical neurons. The protection of neurons from Aβ[31–35] toxicity by PKA inhibition might be mediated by the blocking of the caspase-dependent apoptotic pathways.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Aβ[31–35] induces apoptosis and a significant increase in the activities of caspase-3, -8 and -9 in cortical neurons

To investigate the effects of Aβ[31–35] on neuronal apoptosis, cultured cortical neurons were treated with different concentrations of Aβ[31–35] (0–25 µm) for 24 h, respectively. Incubation of neurons (MAP2-positive cells) with Aβ[31–35] (25 µm) for 24 h induced neuronal apoptosis. Figure 1 presents the data of the ultrastructural studies (A–D: ultrastructure of neurons treated with 25 µm of Aβ[31–35]; E–H: ultrastructure of neurons treated with 100 µm of L-SOP + 25 µm of Aβ[31–35]; and I–L: ultrastructure of neurons treated with 100 µm of (R,S)-PPG + 25 µm of Aβ[31–35]). The neurons in Fig. 1(A,E,I) are the control cells. Figure 1(B–D,F–H,J–L) showed typical neuronal changes of the characteristic apoptosis. Different stages appeared during the apoptotic processes: a patch of condensed chromatin laid against the nuclear membrane, and the shape of the nucleus became irregular, then the nucleus revealed cleavage furrows and finally formed nuclear fragments known as apoptotic bodies. Ultrastructural observations showed that the changes of the characteristic apoptosis in the neurons treated with ‘25 µm of Aβ[31–35]’ did not differ from the changes in the cells exposed to ‘100 µm of L-SOP + 25 µm of Aβ[31–35]’ and ‘100 µm of (R,S)-PPG + 25 µm of Aβ[31–35]’. The treatment of the neurons with 100 µm of L-SOP or (R,S)-PPG significantly reduced the number of apoptotic cells (see the next subsection of Results), but had no significant effect on the ultrastructural changes in the apoptotic neurons.

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Figure 1. Ultrastructural changes in neurons. (A–D) Ultrastructure of neurons treated with 25 µm of Aβ[31–35]. [(A) Control (a normal cell), (B) a patch of condensed chromatin lying against the nuclear membrane and the degenerated organelles, (C) an apoptotic cell was at the center, whose cytoplasm condensed and organelles degenerated and several apoptotic bodies were seen and (D) details of the condensation of cytoplasm and degeneration of organelles.] (E–H) Ultrastructure of neurons treated with 100 µm of L-SOP + 25 µm of Aβ[31–35][(E) Control (a normal cell), (F) patches of condensed chromatin lying against the nuclear membrane, (G) an apoptotic neuron showing the condensed endochylema and organelles and (H) the plasma membrane showing blebbing on the formed apoptosic body)]. (I–L) Ultrastructure of neurons treated with 100 µm of (R,S)-PPG + 25 µm of Aβ[31–35][(I) Control (a normal cell), (J) a degenerated neuron showing depressed nuclear membrane, (K) an apoptotic neuron showing patches of condensed chromatin lying against the nuclear membrane and (L) an apoptotic neuron showing patches of condensed chromatin and condensed endochylema). Scale bars = 1 µm.

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The results obtained from the terminal deoxytransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that the apoptotic cells (%) in the neurons treated without (control) and with Aβ[31–35] (25 µm) were 1.84 ± 0.98 and 26.64 ± 5.76, respectively (P < 0.05) (Fig. 2A,B). The flow cytometric assay showed that the mean values of Annexin–FITC fluorescence intensity in the neurons treated with and without (control) Aβ[31–35] (25 µm) were 28.89 ± 4.27 and 8.69 ± 2.51, respectively (P < 0.05) (Fig. 2C,D). Treatment of the cultured cortical neurons with Aβ[31–35] (25 µm) for 24 h also induced a significant increase in the activities of caspase-3 (Fig. 3A), 8 (Fig. 3B) and -9 (Fig. 3C) in the cortical neurons. The activities of caspase-3, -8 and -9 in the cortical neurons treated with and without (control) Aβ[31–35] (25 µm) were 0.0986 ± 0.0062 and 0.0228 ± 0.0039, 0.0224 ± 0.0021 and 0.0056 ± 0.0011 and 0.0591 ± 0.0046 and 0.0150 ± 0.0027, respectively (all P < 0.05, Aβ[31–35] (25 µm) vs. the control) (Fig. 3).

imageimage

Figure 2. Effects of L-SOP and (R,S)-PPG on Aβ[31–35]-induced apoptosis in primary cortical neurons. The cultured neurons were pretreated with L-SOP (10, 30, 100 µm) or (R,S)-PPG 10, 30, 100 µm for 10 min, and then exposed to 25 µm of Aβ[31–35] for 24 h. (A) A representative terminal deoxytransferase (TdT)-mediated dUTP nick end labeling assay [(a) the Control, (b) 25 µm of Aβ[31–35], (c) 100 µm of L-SOP + 25 µm of Aβ[31–35], (d) 100 µm of (R,S)-PPG + 25 µm of Aβ[31–35])]. (B) Apoptotic cells (%), (C) a representative flow cytometric assay [(a) the control, 25 µm of Aβ[31–35], and 10 µm of L-SOP + 25 µm of Aβ[31–35]; (b) the control, 25 µm of Aβ[31–35], and 30 µm of L-SOP + 25 µm of Aβ[31–35]; (c) the control, 25 µm of Aβ[31–35], and 100 µm of L-SOP + 25 µm of Aβ[31–35]; (d) the control, 25 µm of Aβ[31–35], and 10 µm of (R,S)-PPG + 25 µm of Aβ[31–35]; (e) the control, 25 µm of Aβ[31–35], and 30 µm of (R,S)-PPG + 25 µm of Aβ[31–35]; (f) the control, 25 µm of Aβ[31–35], and 100 µm of (R,S)-PPG + 25 µm of Aβ[31–35]) (blue line: the control, yellow line: 25 µm of Aβ[31–35] and pink line: L-SOP (or (R,S)-PPG) + 25 µm of Aβ[31–35]) and (D) Annexin–FITC fluorescence intensity. Aβ[31–35] (25): 25 µm of Aβ[31–35]; L-SOP (10) (30) or (100): 10, 30 or 100 µm of L-SOP + 25 µm of Aβ[31–35]; and (R,S)-PPG10 (30) or (100): 10, 30 or 100 µm of (R,S)-PPG + 25 µm of Aβ[31–35]. #, P < 0.05 vs. the control; *, P < 0.05 vs. Aβ[31–35] (25).

image

Figure 3. Effects of L-SOP or (R,S)-PPG and/or Sp-cAMP on the activities of caspase-3, -8 and -9 in Aβ[31–35]-treated cortical neurons. The cultured neurons were pretreated with L-SOP (100 µm) or (R,S)-PPG (100 µm) and/or Sp-cAMP (200 µm) for 10 min and then exposed to 25 µm of Aβ[31–35] for 24 h. (A) Caspase-3, (B) caspase-8 and (C) caspase-9. Aβ[31–35]: 25 µm of Aβ[31–35]; Aβ[31–35] + L-SOP (or (R,S)-PPG): 25 µm of Aβ[31–35] + 100 µm of L-SOP (or (R,S)-PPG); Aβ[31–35] + Sp-cAMP: 25 µm of Aβ[31–35] + 200 µm of Sp-cAMP; Aβ[31–35] + L-SOP (or (R,S)-PPG) + Sp-cAMP: 25 µm of Aβ[31–35] + 100 µm of L-SOP (or (R,S)-PPG) + 200 µm of Sp-cAMP. #, P < 0.05 vs. the control; @, P < 0.05 vs. Aβ[31–35]; *, P < 0.05 vs. Aβ[31–35] + L-SOP (or (R,S)-PPG).

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L-SOP and (R,S)-PPG protect neurons from Aβ[31–35]-induced apoptosis and inhibit the effects of Aβ[31–35] on the activities of caspase-3, -8 and -9

To examine the effects of group III mGluR on Aβ[31–35]-induced apoptosis, the cultured cortical neurons were pretreated with one of the two selective agonists, L-SOP (10, 30, 100 µm) or (R,S)-PPG 10, 30, 100 µm, for 10 min and then exposed to Aβ[31–35] (25 µm) for 24 h. Pretreatment of the cells with 10 µm of L-SOP or (R,S)-PPG did not induce any significant changes in the apoptotic cells (%) (Fig. 2A,B) and the Annexin–FITC fluorescence intensity (Fig. 2C,D). However, pretreatment with a higher concentration (30 µm) resulted in a significant decrease in both the apoptotic cells (%) and the Annexin–FITC fluorescence intensity as compared with the Aβ[31–35] (25) group (P < 0.05), and the response of the cells to L-SOP or (R,S)-PPG both peaked at 100 µm[P < 0.05, vs. Aβ[31–35] (25)] in this study.

It was also found that the increased activities of caspase-3 and -9 induced by Aβ[31–35] were almost completely suppressed by the pretreatment of the cortical neurons with L-SOP (100 µm) or (R,S)-PPG (100 µm) (Fig. 3). The activities of caspase-3 (OD 405 nm) in the neurons of the control, L-SOP + Aβ[31–35] and (R,S)-PPG + Aβ[31–35] groups were 0.0228 ± 0.0039, 0.0272 ± 0.0047 and 0.0260 ± 0.0045, respectively, and those of caspase-9 (OD 405 nm) were 0.0150 ± 0.0027, 0.0163 ± 0.0055 and 0.0157 ± 0.0025, respectively (all P > 0.05) (Fig. 3). Pretreatment of the cortical neurons with L-SOP (100 µm) or (R,S)-PPG (100 µm) also induced a significant decrease in the activities of caspase-8 in the Aβ[31–35]-treated neurons [P < 0.05; L-SOP + Aβ[31–35] and (R,S)-PPG + Aβ[31–35] vs. Aβ[31–35]] (Fig. 3).

Effects of PKA activation on indices of neuronal apoptosis induced by Aβ[31–35] and on L-SOP and (R,S)-PPG-induced protection from Aβ[31–35]-induced apoptosis

We next investigated the effects of Sp-cAMP, a potent membrane-permeable activator of cAMP-dependent PKA, on the neuroprotective roles induced by L-SOP or (R,S)-PPG in the Aβ[31–35]-treated neurons. Cultured neurons were pretreated with Sp-cAMP (200 µm) and then exposed to Aβ[31–35] (25 µm) only, L-SOP (100 µm) + Aβ[31–35] (25 µm) or (R,S)-PPG (100 µm) + Aβ[31–35] (25 µm). The data showed that Sp-cAMP has no effect on Aβ[31–35]-induced apoptosis or the increase in the activities of caspase-3, -8 and -9. No significant differences were found in the Annexin–FITC fluorescence intensities (Fig. 4), caspase-3 (Fig. 3A), -8 (Fig. 3B) and -9 (Fig. 3C) between the Aβ[31–35] and Aβ[31–35] + Sp-cAMP groups (all P > 0.05). However, it was found that Sp-cAMP treatment significantly inhibits the effects of L-SOP and (R,S)-PPG on Aβ[31–35]-induced apoptosis and changes in the activities of caspase-3, -8 and -9. Significant differences were found in the Annexin–FITC fluorescence intensity, caspase-3, -8 and -9 between the Aβ[31–35] + L-SOP and Aβ[31–35] + L-SOP + Sp-cAMP groups and between the Aβ[31–35] + (R,S)-PPG and Aβ[31–35] + (R,S)-PPG + Sp-cAMP groups (Figs 3A–C and 4) (all P < 0.05). The results implied that Sp-cAMP has a role in abolishing the neuroprotective effects of group III mGluR agonists on the Aβ[31–35]-treated neurons.

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Figure 4. Effects of L-SOP or (R,S)-PPG and/or Sp-cAMP on Annexin–FITC fluorescence intensity in Aβ[31–35]-treated cortical neurons. The cultured neurons were pretreated with L-SOP (100 µm) or (R,S)-PPG (100 µm) and/or Sp-cAMP (200 µm) for 10 min and then exposed to 25 µm of Aβ[31–35] for 24 h. Aβ[31–35]: 25 µm of Aβ[31–35]; Aβ[31–35] + L-SOP (or (R,S)-PPG): 25 µm of Aβ[31–35] + 100 µm of L-SOP (or (R,S)-PPG); Aβ[31–35] + Sp-cAMP: 25 µm of Aβ[31–35] + 200 µm of Sp-cAMP; Aβ[31–35] + L-SOP (or (R,S)-PPG) + Sp-cAMP: 25 µm of Aβ[31–35] + 100 µm of L-SOP (or (R,S)-PPG) + 200 µm of Sp-cAMP. #, P < 0.05 vs. the control; @, P < 0.05 vs. Aβ[31–35]; *, P < 0.05 vs. Aβ[31–35] + L-SOP (or (R,S)-PPG).

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Rp-cAMP protects neurons from Aβ[31–35]-induced apoptosis and inhibits the effects of Aβ[31–35] on the activities of caspase-3

We also investigated the effects of Rp-cAMP, a specific membrane-permeable inhibitor of cAMP-dependent PKA, on Aβ[31–35]-induced apoptosis and changes in the activities of caspase-3. Cultured neurons were pretreated with Rp-cAMP (10 µm), L-SOP (100 µm) and (R,S)-PPG (100 µm), and then exposed to Aβ[31–35] (25 µm) for 24 h. The findings demonstrated that Rp-cAMP has the ability to almost completely suppress the Aβ[31–35]-induced-apoptosis (Fig. 5A) as well as the increase in the activation of caspase-3 (Fig. 5B). The Annexin–FITC fluorescence intensities (Fig. 5A) and caspase-3 (Fig. 5B) in the Aβ[31–35] + Rp-cAMP group are both significantly lower than those in the Aβ[31–35] group. No significant differences in these measurements were found among the groups of the control, Aβ[31–35] + Rp-cAMP, Aβ[31–35] + L-SOP and Aβ[31–35] + (R,S)-PPG (Fig. 5; all P > 0.05). The findings might imply that Rp-cAMP mimics the neuroprotective effects of group III mGluR agonists on the Aβ[31–35]-treated neurons.

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Figure 5. Effects of L-SOP (R,S)-PPG or Rp-cAMP on Annexin–FITC fluorescence intensity (A) and caspase-3 (B) in Aβ[31–35]-treated cortical neurons. The cultured neurons were pretreated with L-SOP (100 µm) (R,S)-PPG (100 µm) or Sp-cAMP (200 µm) for 10 min and then exposed to 25 µm of Aβ[31–35] for 24 h. Aβ[31–35]: 25 µm of Aβ[31–35]; Aβ[31–35] + Rp-cAMP: 25 µm of Aβ[31–35] + 10 µm of Rp-cAMP; Aβ[31–35] + L-SOP (or (R,S)-PPG): 25 µm of Aβ[31–35] + 100 µm of L-SOP (or (R,S)-PPG). #, P < 0.05 vs. the control; *, P < 0.05 vs. Aβ[31–35].

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Apoptotic cell death associated with the activation of caspases has been reported in several neuronal cell types exposed to AβP (Mattson et al., 1998; Allen et al., 1999, 2001; Troy et al., 2000; Casley et al., 2002; Wei et al., 2002; Lu et al., 2003; Movsesyan et al., 2004; Matsui et al., 2006). In this study, we demonstrated for the first time that Aβ[31–35], a shorter sequence of AβP, induces a significant increase in the activities of not only caspase-3, but also caspase-8 and -9 as well as apoptotic changes including a significant increase in apoptotic cells (%) and the mean Annexin–FITC fluorescence intensity, and the typical and characteristic changes of neuronal apoptosis in the ultrastructural studies. We also found that the activation of group III mGluRs by their agonists L-SOP and (R,S)-PPG provides neuroprotection in the cortical neurons subjected to apoptosis by treatment with Ab[31–35]. Such protection is associated with a significant decrease in the apoptotic cells (%) and the mean Annexin–FITC fluorescence intensity, and the down-regulation of the activities of caspase-3, -8 and -9. Further, we found that Sp-cAMP, an activator of cAMP-dependent PKA, can significantly abolish the L-SOP- and (R,S)-PPG-induced neuroprotection from apoptosis and decrease in caspase-3, -8 and -9 in the Aβ[31–35]-treated neurons. Our findings suggested that neuronal apoptosis induced by Aβ[31–35] is mediated by the PKA-dependent pathway as well as the caspase-dependent intrinsic and extrinsic apoptotic pathways.

The caspases are a family of cysteine proteases and critical mediators of programmed cell death. Caspase-8 and -9 are initiator caspases that are closely linked to pro-apoptotic signals (Ashkenazi & Dixit, 1998; Degterev et al., 2003; Matsui et al., 2006). Caspase-8 is activated by ligand binding to specific death receptors on the cell surface (the extrinsic pathway). Caspase-9 is activated in another apoptotic pathway which emerges from mitochondrial stress (the intrinsic pathway) (Kutuk & Basaga, 2006). These activated upstream caspases then induce an activation of the downstream effector caspases, such as caspase-3, which in their turn cleave cytoskeletal and nuclear proteins to induce apoptosis. Activation of caspase-3 is a key event in the execution of apoptotic cascade in central nervous system diseases including AD. Amyloid β-peptide 31–35-induced activation of caspase-3 has been reported in rat cerebellar granule cells (Misiti et al., 2006). In this study, we found a significant increase in the activities of caspase-3, -8 and -9 as well as the apoptotic changes in the Aβ[31–35]-treated neurons. These implied that the neuronal apoptosis induced by Aβ[31–35] is mediated by the caspase-dependent intrinsic and extrinsic apoptotic pathways. This possibility is further supported by the finding that L-SOP and (R,S)-PPG not only induce a down-regulation of the activities of caspase-3, -8 and -9, but also a significant decrease in the apoptotic cells (%) and the mean Annexin–FITC fluorescence intensity. The finding also suggested that the activation of group III mGluRs protects cortical neurons from Aβ[31–35]-induced apoptosis by blocking the caspase-dependent intrinsic and extrinsic apoptotic pathways.

Both L-SOP and (R,S)-PPG are selective agonists for group III mGluRs with a low micromolar potency (Tanabe et al., 1992; Johansen et al., 1995; Pin & Duvoisin, 1995; Conn & Pin, 1997; Flor et al., 1997; Schoepp et al., 1999). The EC50 values of L-SOP and (R,S)-PPG are about 0.1–7 mm at mGluR4, mGluR6 and mGluR8 except for mGluR7 (Johansen et al., 1995; Conn & Pin, 1997; Flor et al., 1997; Gasparini et al., 1999). However, our data showed that 10 µm of (R,S)-PPG and L-SOP did not induce any significant changes in the apoptotic cells (%) and Annexin–FITC fluorescence intensity. The reasons responsible for this unexpected result are unknown. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b or hmGluR8a, Gasparini et al. (1999) reported that the EC50 values for (R,S)-PPG are 5.2 ± 0.7 µm, 4.7 ± 0.9 µm, 185 ± 42 µm and 0.2 ± 0.1 µm, respectively. This shows that (R,S)-PPG has a higher potency for mGlu8a than mGlu6 or mGlu4a, and the lowest potency at mGlu7b. In the four cell lines expressing recombinant group III mGluRs, they found that the extent of maximal inhibition of cAMP formation by (R,S)-PPG varied, ranging from 45% to 80% of inhibition (Gasparini et al., 1999). This demonstrated that (R,S)-PPG (and L-SOP) have different affinities for the individual group III mGluRs, and the potency of (R,S)-PPG (and L-SOP) for group III mGluRs could be different in different cell lines or cell cultures under different experimental conditions. In the present study, Aβ[31–35] might have the ability to induce a decrease in the potency of (R,S)-PPG and L-SOP for group III mGluRs under our experimental conditions. It is probably one of the reasons why 10 µm of (R,S)-PPG and L-SOP has no effect on the apoptotic cells (%) and Annexin–FITC fluorescence intensity, while 30 µm induces a significant decrease in them. This possibility needs to be further confirmed.

The cAMP–PKA pathway has been reported to be associated with the physiopathology of AD (Martinez et al., 1999), Aβ[1–42] toxicity in primary neuronal cultures (Echeverria et al., 2005), allicin-induced cell death (Park et al., 2005) and anisomycin-induced apoptosis (Chiarini et al., 2002). Therefore, we also investigated the effects of the inhibitor or activator of the cAMP-dependent PKA on apoptotic indices and caspase activities in cortical neurons treated with Aβ[31–35] or cotreated with L-SOP or (R,S)-PPG and Aβ[31–35]. Rp-cAMP (an inhibitor) was found to have the ability to significantly protect neurons from Aβ[31–35]-induced apoptosis and to reverse almost completely the effects of Aβ[31–35] on the activities of caspase-3. This finding provides strong evidence for the involvement of a PKA-dependent pathway in the development of apoptosis in the Aβ[31–35]-treated neurons. The inhibitory ability of Rp-cAMP on caspase-3 suggests a functional association between PKA and the caspase-dependent pathway. Rp-cAMP probably protects neurons by first inhibiting PKA and then the caspase-dependent apoptotic pathway.

Although Rp-cAMP (an inhibitor) induced a significant decrease in the activities of caspase-3, Sp-cAMP (an activator) did not produce any significant increase in the level of caspases in the Aβ[31–35]-treated neurons. There were no significant differences in the level of caspases of the neurons between the Aβ[31–35] and Sp-cAMP + Aβ[31–35] groups. Theoretically, an activator of PKA should induce an increase in the level of caspases if the PKA has a functional association with the caspase-dependent pathway as we suggested. However, the finding obtained is not as we expected. This is likely because of most, if not all, of the PKAs having already been activated by Aβ[31–35], and thus the levels of caspases are already much higher in the Aβ[31–35]-treated neurons under our experimental conditions. The treatment of neurons with an activator of PKA is therefore not able to induce any further increase in the level of caspases.

Sp-cAMP has no effect on the level of caspases in the Aβ[31–35]-treated neurons. However, this activator can significantly revise the effects of L-SOP and (R,S)-PPG on neuronal apoptosis and caspase-3, -8 and -9 in the Aβ[31–35]-treated neurons. Both the activities of caspase-3, -8 and -9 and the mean Annexin–FITC fluorescence intensity are significantly higher in the neurons of the Aβ[31–35] + L-SOP + Sp-cAMP and Aβ[31–35] + (R,S)-PPG + Sp-cAMP groups than those in the Aβ[31–35] + L-SOP and Aβ[31–35] + (R,S)-PPG groups (All P < 0.05). These observations provide further evidence for the functional connection between the caspase- and the PKA-dependent pathways in Aβ[31–35]-induced apoptosis in cortical neurons. The increase in the activities of caspase-3, -8 and -9 and the mean Annexin–FITC fluorescence intensity in the neurons of the Aβ[31–35] + L-SOP + Sp-cAMP and Aβ[31–35] + (R,S)-PPG + Sp-cAMP groups is probably caused by the increased PKA activities induced by Sp-cAMP. The increased PKA is then able to stimulate the caspase-dependent intrinsic and extrinsic apoptotic pathways, leading to neuronal apoptosis.

In summary, our findings implied that neuronal apoptosis induced by Aβ[31–35] is mediated by the caspase-dependent intrinsic and extrinsic apoptotic pathways as well as the PKA-dependent pathway. Amyloid β-peptide 31–35 might have the ability to activate PKA first, which in turn activates the caspase-dependent intrinsic and extrinsic apoptotic pathways, inducing apoptosis in cortical neurons. The data also demonstrated that the activation of group III mGluRs and the inhibition of cAMP-dependent PKA protect cortical neurons from Aβ[31–35]-induced apoptosis by blocking the caspase-dependent apoptotic pathways, suggesting a potential therapeutic role for group III mGluR agonists and inhibitors of cAMP-dependent PKA in the treatment of Aβ-induced neuronal degeneration.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Materials

Mouse monoclonal anti-MAP2, Aβ[31–35], Sp-cAMP, Rp-cAMP, Annexin V–FITC kit, poly l-lysine, FBS, glutamine and d-Hank's were purchased from Sigma Chemical Co., St Louis, MO, USA. Dulbecco's modified Eagle's medium and FBS were from Gibco-BRL, Carlsbad, CA, USA. Caspases (including caspase-3, caspase-8 and caspase-9) colorimetric protease assay kit was purchased from the United States Biological, Swampscott, MA, USA, and TUNEL assay kit from Boehringer, Sandhoferstr, Mannheim, Germany. l-Serine-O-phosphate and (R,S)-PPG were obtained from Tocris Neuramin, Essex, UK and dissolved in d-Hank's at an initial concentration of 25 mm and stored at –20 °C. Amyloid β-peptide 31–35 was dissolved in DMEM at an initial concentration of 2.5 mm, stored at –20 °C and applied at a concentration of 5–25 µm. Sp-cAMP or Rp-cAMP was dissolved in double distilled water at a concentration of 25 mm or 1 mm and stored at –20 °C.

Primary cell culture

Primary cortical neurons were cultured and identified with MAP2 as described previously (Ho et al., 2003). In brief, the prefrontal cortex was aseptically removed from 1- to 3-day-old SD rats, minced with sterile surgical blades, incubated in 0.125% trypsin and dissociated by trituration in DNase/trypsin inhibitor solution. Dissociated cortical cells were diluted in DMEM/Ham's F12 medium (1 : 1, v/v, pH 7.2) containing 10% heart-inactivated FBS glutamine (4 mm), 4.5 g L−1 d-glucose and penicillin (100 units), and then plated on poly l-lysine-coated coverslips. Non-neuronal cell division was inhibited by exposure to cytarabine (Sigma) for 24 h. The cultures were maintained at 37 °C in a humidified environment with 5% CO2 in a CO2 incubator (TC2323). After 10 days in culture (10 days in vitro, DIV), observation through a phase-contrast microscope demonstrated that the cells were predominantly neuronal cells (> 98%). The cells at this stage were used in the experiment.

Experimental design

To investigate the effects of Aβ[31–35] on neuronal apoptosis, cultured cortical neurons were treated with different concentrations of Aβ[31–35] (0, 5, 10, 20, 25 µm) for 24 h, respectively. To determine the effects of group III mGluR on Aβ[31–35]-induced apoptosis, the cultured cortical neurons were pretreated with one of the two selective agonists, L-SOP (10, 30, 100 µm) or (R,S)-PPG 10, 30, 100 µm for 10 min, and then incubated with Aβ[31–35] (25 µm) for 24 h. To examine whether the cAMP–PKA pathway was involved in the Aβ[31–35]-induced apoptosis, the cortical neurons were pretreated with or without Sp-cAMP (200 µm), a potent membrane-permeable activator of cAMP-dependent PKA or Rp-cAMP (10 µm), a specific membrane-permeable inhibitor of cAMP-dependent PKA for 10 min, and then with or without Aβ[31–35] (25 µm) and L-SOP (100 µm) or (R,S)-PPG (100 µm) for 24 h. After the mentioned treatments, morphological analysis, flow cytometric assay, TUNEL examinations and/or the measurements of activities of caspases (caspase-3, -8 and -9) were conducted.

Ultrastructural studies

Cultured cortical neurons treated with 25 µm of Aβ[31–35] for 24 h were fixed with 2% glutaraldehyde for 2 h and then 1% buffered OsO4 for 2 h. The cultures were then dehydrated with graded alcohols and embedded in Epon 618, polymerized at 60 °C for 3 days. The thin sections were stained with uranyl acetate and lead citrate, and viewed with JEOL 100-CX transmission electron microscopy (JEOL Ltd., Tokyo, Japan).

Flow cytometric assay

Univariate flow cytometric measurements were performed using an FACS 440 flow cytometer (Becton, Dickinson, Frankin Lakes, NJ, USA) with argon ion laser excitation at 488 nm beam. Red fluorescene (DNA) was detected through a 563–607 nm bandpass filter. Ten thousand cells in each sample were analyzed. The data were analyzed using a single histogram statistics software. The cells were stained with Annexin V–FITC and PI for 20 min. The Annexin V–FITC-positive cells were early apoptotic cells, which were described by the mean fluorescent intensity.

TUNEL assay

Apoptotic cell death was quantified by DNA strand breaks which were detected in situ by the TUNEL method. In brief, cultures were fixed in fresh 4% paraformaldehyde solution (PBS, pH 7.4), permeabilized with 0.1% Triton X-100 at room temperature for 15 min. After quenching endogenous peroxidase in 3.0% hydrogen peroxide, the cultures were treated with TdT enzyme in a humidified chamber at 37 °C for 1 h. The cultures were incubated with HRPase-conjugated anti-digoxigenin antibody in a humidified chamber at room temperature for 30 min, and then developed color with diaminobenzidine. Apoptotic cells could be discriminated morphologically by the presence of condensed brown nuclei. The number of the TUNEL-positive cells and the total number of cells were counted. A minimum of 400 cells were counted for each control and experimental data point.

Measurement of caspase activity

Caspase activity was measured by using a United States Biological assay kit following the manufacturer's instructions. DEVD–p-nitroanilide (pNA) was used as a colorimetric substrate. Primary cultured cortical neurons were plated at a density of 1 × 106 cells per culture dish. After different treatments as designed, the cells were harvested by centrifugation. The pellets were washed with PBS and lysed in 50 µL of chilled cell lysis buffer and incubated in ice for 10 min. Lysate was centrifuged at 10 000 r.p.m. for 1 min at 4 °C, and the supernatant was used for the caspase-3, -8 and -9 assay. The protein concentration was confirmed by the bicinchoninic acid assay. The protease activity was determined by the spectrophotometric detection at 405 nm of the chromophore pNA after its cleavage by caspases from the labeled caspases-specific substrate (DEVD–pNA). Background reading from cell lysates and buffers was subtracted from the readings of both induced and uninduced samples before calculating the fold increase in the caspase activity.

Statistical analysis

Data are expressed as means ± standard error of mean. All statistical evaluations were performed using two-tailed Student's t-test, and data were analyzed using the computer program spss (version 11.0). A probability level of P < 0.05 was considered to be significant.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

This work was supported by research grants from Shanxi Medical University, Chinese University of Hong Kong (Direct grant Project: 4450226) and HK Polytechnic University (Niche Area).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
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