Elsevier

Experimental Neurology

Volume 184, Issue 2, December 2003, Pages 923-929
Experimental Neurology

A three amino acid peptide, Gly-Pro-Arg, protects and rescues cell death induced by amyloid β-peptide

https://doi.org/10.1016/S0014-4886(03)00314-5Get rights and content

Abstract

Amyloid β-peptide (Aβ) contributes to the pathogenesis of Alzheimer's disease (AD), causing neuronal death through apoptosis. In this study, the neuroprotective role of small peptides, Gly-Pro-Glu (GPE), Gly-Glu (GE), Gly-Pro-Asp (GPD), and Gly-Pro-Arg (GPR) were examined against Aβ-induced toxicity in cultured rat hippocampal neurons. We report here that GPR (10–100 μM) prevented Aβ-mediated increase in lactate dehydrogenase (LDH) release and Aβ inhibition of MTT reduction, even in neurons that were pre-exposed to Aβ for 24 or 48 h. Since GPR prevented Aβ inhibition of MTT reduction, the anti-apoptotic effect of GPR was studied by examining activation of caspase-3 and expression of p53 protein. Caspase-3 was significantly activated by 20 μM Aβ25–35 and 5 μM Aβ1–40, but GPR effectively prevented the Aβ-mediated activation of caspase-3. Similarly, Aβ increased numbers of p53-positive cells, but GPR prevented this Aβ effect. Our findings suggest that GPR can rescue cultured rat hippocampal neurons from Aβ-induced neuronal death by inhibiting caspase-3/p53-dependent apoptosis.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with dementia as a major clinical symptom. The brain of AD patients is characterized by deposition of amyloid β-peptide (Aβ), a 39- to 43-amino acid peptide (Yankner, 1996). Although Aβ has been implicated as a cause of neuronal degeneration (Mattson, 1997), the precise mechanism of its neurotoxic action remains unclear. Post-mortem AD brains have shown a large number of neurons with fragmented DNA Dragunow et al., 1995, Lassmann et al., 1995, Masliah et al., 1998. Although not all neurons showed other morphological features of apoptosis, for example, cell surface blebbing, cell shrinkage, and nuclear condensation Lassmann et al., 1995, Lucassen et al., 1987, Stadelmann et al., 1998, neurons did show altered expression of apoptosis-related proteins, such as caspase-3, caspase-8, caspase-9 Lu et al., 2000, Rohn et al., 2001, Par-4, bak, bad, bax, bcl-2, p53, CPP32, and fas Guo et al., 1998, Kitamura et al., 1997, Kitamura et al., 1998, Nishimura et al., 1995, Shinohara et al., 1999, Stadelmann et al., 1999, Su et al., 1996, Su et al., 1997. What causes neuronal apoptosis in the brain of AD patients has not been determined; however, Aβ induces apoptosis in cultured neurons Gschwind and Huber, 1995, Li et al., 1996 and inhibition of p53, a key regulatory molecule of apoptosis, was shown to prevent Aβ-induced neuronal death (Culmsee et al., 2001). These in vivo and in vitro observations suggest that Aβ induces apoptosis in the brain of AD patients.

Studies of the mechanisms whereby Aβ induces neuronal degeneration have led to the identification of compounds capable of protecting neurons. They include peptide aggregation-blocking agents Permanne et al., 2002, Soto et al., 1998, neurotrophic factors (Guo and Mattson, 2000), antioxidants Behl et al., 1992, Goodman and Mattson, 1994, Kumar et al., 1994, drugs that affect calcium signals (Weiss et al., 1994), and estrogens (Goodman and Mattson, 1994). Several proteins and peptides were also reported to prevent Aβ-induced neuronal degeneration. They include tachykinin neuropeptides (substance P) (Yankner et al., 1990), vasoactive intestinal peptide (VIP) (Gozes et al., 1999), and activity-dependent neuroprotective protein (ADNP; i.e., VIP fragment of either 8 or 14 amino acids) Brenneman and Gozes, 1996, Zemylak et al., 2000, and IGF-1 (Doré et al., 1997). Interestingly, peptide Gly-Pro-Glu (GPE), a product of IGF-I, was shown to protect against NMDA-induced toxicity (Saura et al., 1999), hypoxic–ischemic injury (Sizonenko et al., 2001), and 6-OHDA-induced nigral lesions (Guan et al., 2000). In this study, we examined the neuroprotective action of small peptides, GPE, Gly-Glu (GE), Gly-Pro-Asp (GPD), and Gly-Pro-Arg (GPR) against Aβ toxicity. GPR, but not other peptides, effectively prevented neuronal degeneration by preventing Aβ-induced activation of caspase-3 and increased expression of p53, suggesting that GPR prevents Aβ-mediated apoptosis.

Section snippets

Cell culture and Aβ peptide

Hippocampal neurons were obtained from 17E Sprague–Dawley rats. Cell suspension with a density of 1 to 3 × 105 cells/ml in neurobasal media with B27 supplement (Life Technologies) was plated in 24-well tissue culture plates coated with polyethylenimine (PEI). Cultures were incubated at 37°C in 5% CO2-humidified atmosphere. All pups from each pregnant rat are used as an experimental unit (n). Aβ25–35 and Aβ1–40 (Bachem) were dissolved in deionized water at a concentration of 6 mg/ml and stored

GPR prevents Aβ-mediated increase in LDH

Neuronal death, cultured rat hippocampal neurons were exposed to Aβ25–35 with or without peptides. Aβ-induced increase in LDH levels was about 70% greater than that in the control culture (Fig. 1). Three peptides, GE (50 μM), GPD (50 μM), and GPE (50 and 100 μM) did not prevent Aβ-mediated LDH increase (data not shown). However, GPR (10 and 100 μM) was effective in preventing Aβ-mediated increase in LDH levels [Fig. 1; F(2,36) = 4.5, P = 0.02]. When GPR was added together with Aβ or even added

Discussion

It was shown that 75% of hippocampal neurons die after 6-day incubation with Aβ25–35 (25–50 μM) (Lockhart et al., 1994). The present study demonstrated that Aβ-induced neurotoxicity can be protected by GPR. Most importantly, LDH and MTT assays suggest that GPR effectively rescues neurons that have been pre-exposed to aggregated Aβ for up to 2 days. Similar findings have been reported for IGF-I (Doré et al., 1997). Furthermore, GPE, a peptide structurally identical to the N-terminal tripeptide

Acknowledgements

The authors thank Cathy Martens for her technical support.

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    This study was supported by a grant from the Alzheimer's Association (IIRG-00-2189).

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