Protective effect of apolipoprotein E-mimetic peptides on N-methyl-d-aspartate excitotoxicity in primary rat neuronal–glial cell cultures

doi:10.1016/S0306-4522(02)00709-1

Neuroscience

Volume 116, Issue 2, 31 January 2003, Pages 437-445

https://doi.org/10.1016/S0306-4522(02)00709-1 Get rights and content

Abstract

Apolipoprotein E (apoE) is a 34-kD protein with multiple biological properties. Recent clinical and preclinical observations implicate a role for apoE in modifying the response of the brain to focal and global ischemia. One mechanism by which apoE might exert these effects is by reducing glutamate-induced excitotoxic neuronal injury associated with ischemic insults. We demonstrate that human recombinant apoE confers a mild neuroprotective effect in primary neuronal–glial cultures exposed to 100 μM N-methyl-d-aspartate. Furthermore, a peptide derived from the receptor-binding region of apoE (residues 133-149) maintained a significant helical population as assessed by circular dichroism, and completely suppressed the neuronal cell death and calcium influx associated with N-methyl-d-aspartate exposure. Neuroprotection was greatest when the peptide was added concurrently with N-methyl-d-aspartate; however, a significant protection was observed when peptide was preincubated and washed off prior to N-methyl-d-aspartate exposure.

These results suggest that one mechanism by which apoE may modify the CNS response to ischemia is by partially blocking glutamate excitotoxicity. Moreover, small peptide fragments derived from the receptor-binding region of apoE have enhanced bioactivity compared with the intact holoprotein, and may represent a novel therapeutic strategy for the treatment of brain ischemia.

Section snippets

Experimental procedures

All animal procedures were designed to minimize animal discomfort and numbers, and were approved by the Duke University Animal Care and Use Committee.

Results

To investigate the ability of apoE to protect cells from glutamate excitotoxicity in an experimental tissue culture model of cerebral ischemia, primary rat neuronal–glial cultures were preincubated with human recombinant apoE prior to exposure of the cells to NMDA. Previous experiments performed in our laboratory failed to detect an isoform specific effect of human recombinant apoE on NMDA-induced excitotoxicity in primary rodent neuronal–glial cultures (Aono et al., 2002). To this end apoE3,

Discussion

In this study, we demonstrate that peptide sequences derived from the receptor-binding region of apoE exert a protective effect against NMDA-mediated neuronal excitotoxicity in a tissue culture model of cerebral ischemia. This neuroprotective effect of the apoE peptide was both specific and dose-dependent. At a concentration of 6 μ a 17-residue peptide, apoE (133–149) blocked the neurotoxicity and calcium influx associated with exposure of primary neuronal–glial cultures to 100 μM NMDA as

Acknowledgements

Financial support for this study was provided by NIH 1K08NS01949, R01NS37235, 1R01NS368087, and the Paul Beeson Physician Faculty Awards (D.T.L.).

References (45)

M.J. Alberts et al.
ApoE genotype and survival from intracerebral haemorrhage
Lancet
(1995)
D.H. Kim et al.
Human apolipoprotein E receptor 2
J Biol Chem
(1996)
A. Lalazar et al.
Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions
J Biol Chem
(1988)
D.T. Laskowitz et al.
Downregulation of microglial activation by Apolipoprotein E and apoE-mimetic peptides
Exp Neurol
(2001)
J.R. Lynch et al.
Apolipoprotein E modulates glial activation and the endogenous central nervous system inflammatory response
J Neuroimmunol
(2001)
B. Meldrum et al.
Excitatory amino acid neurotoxicity and neurodegenerative disease
Trends Pharmacol Sci
(1990)
S. Novak et al.
A new low density lipoprotein receptor homologue with 8 binding ligand repeats in brain of chicken and mouse
J Biol Chem
(1996)
B.E. Tardiff et al.
Preliminary report of a genetic basis for cognitive decline after cardiac operations. The Neurologic Outcome Research Group of the Duke Heart Center
Ann Thorac Surg
(1997)
G.M. Teasdale et al.
Association of apolipoprotein E polymorphism with outcome after head injury
Lancet
(1997)
K.H. Weisgraber et al.
Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site
J Biol Chem
(1982)

K.H. Weisgraber

Apolipoprotein Estructure-function relationships

Adv Protein Chem

(1994)

T. Wisniewski et al.

Apolipoprotein Ea pathological chaperone in patients with cerebral and systemic amyloid

Neurosci Let

(1992)

E. Amador et al.

Serum lactate dehydrogenase activityan analytical assessment of current assays

Clin Chem

(1963)

Aono M, Lee Y, Zivin RA, Pearlstein RD, Warner DS, Bennett ER, Laskowitz DT (2002). Apolipoprotein E protects against...

B.J. Bacskai et al.

The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-d-aspartate receptors

Proc Natl Acad Sci USA

(2000)

R.D. Bart et al.

Regional cerebral blood flow in apolipoprotein E deficient vs wildtype mice undergoing middle cerebral artery occlusion

Neuroreport

(1998)

J. Broderick et al.

Apolipoprotein E phenotype and the efficacy of intravenous tissue plasminogen activator in acute ischemic stroke

Ann Neurol

(2001)

E.H. Corder et al.

Gene doses of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families

Science

(1993)

G. Friedman et al.

Apolipoprotein E-epsilon 4 genotype predicts a poor outcome in survivors of traumatic brain injury

Neurology

(1999)

D.M. Holtzman et al.

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line

Proc Natl Acad Sci USA

(1995)

K. Horsburgh et al.

Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia

Neuroreport

(1999)

K. Horsburgh et al.

Intraventricular infusion of apolipoprotein E ameliorates acute neuronal damage after global cerebral ischemia in mice

J Cereb Blood Flow Metab

(2000)

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Research paperProtective effect of apolipoprotein E-mimetic peptides on N-methyl-d-aspartate excitotoxicity in primary rat neuronal–glial cell cultures

Abstract

Section snippets

Experimental procedures

Results

Discussion

Acknowledgements

Lancet

J Biol Chem

J Biol Chem

Exp Neurol

J Neuroimmunol

Trends Pharmacol Sci

J Biol Chem

Ann Thorac Surg

Lancet

J Biol Chem

Adv Protein Chem

Neurosci Let

Serum lactate dehydrogenase activityan analytical assessment of current assays

Clin Chem

The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-d-aspartate receptors

Proc Natl Acad Sci USA

Regional cerebral blood flow in apolipoprotein E deficient vs wildtype mice undergoing middle cerebral artery occlusion

Neuroreport

Apolipoprotein E phenotype and the efficacy of intravenous tissue plasminogen activator in acute ischemic stroke

Ann Neurol

Gene doses of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families

Science

Apolipoprotein E-epsilon 4 genotype predicts a poor outcome in survivors of traumatic brain injury

Neurology

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line

Proc Natl Acad Sci USA

Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia

Neuroreport

Intraventricular infusion of apolipoprotein E ameliorates acute neuronal damage after global cerebral ischemia in mice

J Cereb Blood Flow Metab

Research paper
Protective effect of apolipoprotein E-mimetic peptides on N-methyl-d-aspartate excitotoxicity in primary rat neuronal–glial cell cultures