Effects of positive AMPA receptor modulators on calpain-mediated spectrin degradation in cultured hippocampal slices

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Abstract

Positive modulators of AMPA receptors (AMPAr), also known as ampakines, are allosteric effectors of the receptors and have been extensively studied in past years due to their potential use as treatment for various diseases and ailments of the central nervous system such as mild cognitive impairment, schizophrenia, and Alzheimer's disease. Ampakines have been shown to improve performance on memory tasks in animals and in human subjects, an effect linked to their ability to increase agonist-mediated ion influx through AMPAr, thus leading to enhanced synaptic responses and facilitation of long-term potentiation (LTP) induction at glutamatergic synapses. As LTP is associated with calpain activation and spectrin degradation, we determined the effects of ampakine treatment of cultured hippocampal slices on spectrin degradation. Calpain activation was evaluated by determining the levels of the 145–150 kDa degradation products of spectrin. Our data indicated that incubation of hippocampal slices with some, but not all positive modulators of AMPA receptors resulted in enhanced spectrin degradation, an effect that was blocked by a calpain inhibitor. In addition, an antagonist of AMPAr but not of NMDAr blocked ampakine-induced spectrin degradation. These results indicate that prolonged treatment with selected ampakines leads to spectrin degradation mediated by activation of the calcium-dependent protease calpain.

Introduction

Calpain is a neutral Ca2+-activated cysteine protease present in postsynaptic structures where it has a wide range of substrates (Lynch and Baudry, 1987, Goll et al., 2003). This protease is essential for the induction of synaptic long-term potentiation (LTP) (Oliver et al., 1989, Muller et al., 1995, Bednarski et al., 1995, Vanderklish et al., 1996, Caba et al., 2002). One of the preferred calpain substrates in brain is brain spectrin; brain spectrin, initially referred to as fodrin, is a prominent component of the membrane-associated cytoskeleton. Substantial evidence implicates spectrin in the regulation of cellular morphology in red blood cells and muscle sarcomeres, and in the clustering of acetylcholine receptors at neuromuscular junctions (Bloch and Morrow, 1989). Distinct isoforms of spectrin are expressed in neurons (Hesketh et al., 1983), and calpain-mediated spectrin proteolysis has been associated with marked and lasting effects on the morphology and physiology of synapses (Lynch and Baudry, 1987, Bednarski et al., 1995). Calpain-mediated spectrin degradation results in the formation of 145–150 kDa breakdown products, and represents site-specific and activity-dependent markers of synaptic activity (Lynch and Baudry, 1987, Saido et al., 1993, Dosemeci and Reese, 1995, Vanderklish et al., 2000, Zakharov and Mosevitsky, 2001). It has been proposed that calpain activation and spectrin degradation could reflect rapid and long lasting reorganizations of postsynaptic structures under physiological or pathological conditions (Masliah et al., 1990, Lee et al., 1991, Vanderklish and Bahr, 2000, Neumar et al., 2001, Caba et al., 2002).

Glutamate release in the brain elicits a fast postsynaptic response mediated by the opening of AMPA-type glutamate receptor (AMPAr) channels. This response depends on binding of glutamate to the receptor and can be positively modulated by a group of allosteric effectors of these receptors called ampakines. Ampakines have been shown to bind to the AMPA receptors at a site distinct from the glutamate site and to increase AMPAr-mediated current in acute and cultured hippocampal slices and in vivo, to improve performance on several behavioral tasks when administered to animals, and to enhance memory encoding in humans (Ingvar et al., 1997; Hampson et al., 1998a, Hampson et al., 1998b; Arai et al., 2000, Suppiramaniam et al., 2001). Furthermore, several ampakines have been shown to facilitate LTP induction in hippocampal slices and in vivo, and this effect has been linked to their cognitive enhancing properties. In view of the proposed role of calpain in LTP induction, it was of interest to determine the effect of ampakines on calpain activation and spectrin degradation in hippocampus. Our results indicate that treatment with some, but not all, positive modulators of AMPA receptors results in calpain activation and spectrin degradation.

Section snippets

Hippocampal slice culture

Ten to 13 day-old rat pups were obtained from Jackson Laboratories (Bar Harbor, Maine) and used to prepare hippocampal slice cultures as previously described (Stoppini et al., 1991). Briefly, animals were decapitated and the brains were dissected, trimmed, and 400 μm thick hippocampal slices were cut using a McIllwain tissue slicer (Fotodyne Inc., New Berlin, WI). Slices were plated onto Millipore insert membranes, placed in six-well culture plates, and fed with regular slice culture medium (50%

Treatment of cultured hippocampal slices with CX614 increases spectrin degradation

Calpain is activated by increased intracellular Ca2+ concentrations, and calpain-mediated spectrin degradation specifically produces a doublet band of 145–150 kDa. We monitored this characteristic band as an indicator of calpain activation. Brain membrane fractions treated with 2 mM CaCl2 (see Section 2) were used as a positive control for calpain-mediated spectrin degradation (Massicotte et al., 1990, Lu et al., 2001). Hippocampal slices were incubated with the ampakine CX614 (50 μM for 48 h) or

Discussion

The present results indicate that treatment of cultured hippocampal slices with some but not all positive AMPA receptor modulators was associated with relatively rapid and sustained calpain activation, as evidenced by the accumulation of spectrin breakdown products identical to those generated by calpain-mediated spectrin degradation (Siman et al., 1984, Bahr et al., 1995, Bi et al., 1996). In addition, a calpain inhibitor prevented the accumulation of spectrin breakdown products. CX614 is a

Acknowledgements

We would like to thank Dr. Jihua Liu and Miss Gabriela Saavedra for excellent technical assistance. This research was supported by Grant P01NS045260-01 (PI: Dr. C.M. Gall) from NINDS.

References (59)

  • J.E. Hesketh et al.

    Evidence for a spectrin-like protein as a major component of the synaptosomal membrane cytoskeleton

    Biochem. Biophys. Res. Commun.

    (1983)
  • M. Ingvar et al.

    Enhancement by an ampakine of memory encoding in humans

    Exp. Neurol.

    (1997)
  • Y. Iwakura et al.

    N-methyl-d-aspartate-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor down-regulation involves interaction of the carboxyl terminus of GluR2/3 with Pick1. Ligand-binding studies using Sindbis vectors carrying AMPA receptor decoys

    J. Biol. Chem.

    (2001)
  • R.J. Knapp et al.

    Antidepressant activity of memory-enhancing drugs in the reduction of submissive behavior model

    Eur. J. Pharmacol.

    (2002)
  • B. Lockhart et al.

    (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPA receptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rat hippocampal and frontal cortex slices

    Eur. J. Pharmacol.

    (2000)
  • X. Lu et al.

    Calpain-mediated degradation of PSD-95 in developing and adult rat brain

    Neurosci. Lett.

    (2000)
  • G. Lynch et al.

    Brain spectrin, calpain and long-term changes in synaptic efficacy

    Brain Res. Bull.

    (1987)
  • E. Masliah et al.

    Increased immunoreactivity of brain spectrin in Alzheimer disease: a marker for synapse loss?

    Brain Res.

    (1990)
  • S. Marenco et al.

    Preliminary experience with an ampakine (CX516) as a single agent for the treatment of schizophrenia: a case series

    Schizophr. Res.

    (2002)
  • S. Munirathinam et al.

    Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors elicits neuroprotection after trimethyltin exposure in hippocampus

    Toxicol. Appl. Pharmacol.

    (2002)
  • S. Murase et al.

    Depolarization drives beta-Catenin into neuronal spines promoting changes in synaptic structure and function

    Neuron

    (2002)
  • N. Nagarajan et al.

    Mechanism and impact of allosteric AMPA receptor modulation by the ampakine CX546

    Neuropharmacology

    (2001)
  • I. Najm et al.

    Changes in polyamine levels and spectrin degradation following kainate-induced seizure activity: effect of difluoromethylornithine

    Exp. Neurol.

    (1992)
  • R.W. Neumar et al.

    Calpain activity in the rat brain after transient forebrain ischemia

    Exp. Neurol.

    (2001)
  • M.W. Oliver et al.

    The protease inhibitor leupeptin interferes with the development of LTP in hippocampal slices

    Brain Res.

    (1989)
  • T.C. Saido et al.

    Spatial resolution of fodrin proteolysis in postischemic brain

    J. Biol. Chem.

    (1993)
  • L. Stoppini et al.

    A simple method for organotypic cultures of nervous tissue

    J. Neurosci. Meth.

    (1991)
  • P. Vanderklish et al.

    Proteolysis of spectrin by calpain accompanies theta-burst stimulation in cultured hippocampal slices

    Brain Res. Mol. Brain Res.

    (1995)
  • V.V. Zakharov et al.

    Site-specific calcium-dependent proteolysis of neuronal protein GAP-43

    Neurosci. Res.

    (2001)
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