Transforming growth factors-β protect primary rat hippocampal neuronal cultures from degeneration induced by β-amyloid peptide

doi:10.1016/0006-8993(96)00458-1

Brain Research

Volume 732, Issues 1–2, 2 September 1996, Pages 16-24

https://doi.org/10.1016/0006-8993(96)00458-1 Get rights and content

Abstract

Treatment of primary rat embryo hippocampal neuronal cultures with 10⁻⁵ M β-amyloid peptide fragment 25–35 (AβP) for 24 h resulted in a 60% decrese in cell viability as determined by MTT incorporation. When these cells were treated with 0.1–10 ng/ml of either transforming growth factor-β (TGF-β) 1, 2 or 3 for 24 h before exposure to AβP, there was a 2.9-, 1.9-, and 3.2-fold increase in cell survival, respectively, compared to cells treated with AβP alone. The viability of cells treated with AβP and 0.1–10 ng/ml TGF-β was comparable to that of cells not treated with AβP. The protective effects were less pronounced at lower TGF-β concentrations. The protective effects of pretreatment with TGF-β were less striking in mouse CCL-N-2a and human SK-N-SH neuroblastoma cell lines. When all cells were treated with TGF-β for 24 h following a 24 h exposure to AβP, there was a trend toward increased cell viability which was less significant than pretreatment with TGFs-β. An isoform-specific TGF-β SELISA showed that primary hippocampal neuronal cultures and the neuroblastoma cell lines secrete all 3 TGF-β isoforms. Based on our results, we propose that the increased expression of TGF-β observed in brains of patients with Alzheimer's disease may offer some degree of neuroprotection.

Introduction

Alzheimer's disease (AD) is one of the major neurodegenerative diseases of aging. The neuropathological hallmarks of AD are senile plaques and neurofibrillary tangles. In vitro studies have shown that aggregates of β-amyloid peptide (AβP) directly induce neuronal loss, as well as dystrophic neurite morphology in primary neuronal cultures 2, 28, 29, 30, 31. Neuronal degeneration has also been shown in adult rats and aged primates following intracerebral injection of β-amyloid plaque cores and AβP aggregates 10, 18, 19, 36, 40. Recently a transgenic mouse expressing high levels of human mutant amyloid precursor proteins has been shown to exhibit much of the pathology of AD [11].

Transforming growth factors-β (TGFs-β) are multifunctional peptide growth factors that regulate the processes of proliferation and differentiation in many types of cells. Their actions depend not only on the type of cell and its differentiation state, but also on growth conditions and the presence of other biological signalling molecules 23, 35. Mammals express three highly homologous isoforms (TGF-β 1–3) which share many biological activities in vitro, but which seem to have differential effects in the central nervous system 8, 9. TGFs-β 2 and 3 have been localized in a wide range of neuronal and glial populations in the central nervous system of rats and mice 8, 37, while TGF-β1 is more often expressed following injury 17, 21, 26. Recently, in vitro and in vivo studies suggest that TGF-β1 protects against injury in cells of the central nervous system. In vitro, TGF-β1 decreases neuronal degeneration caused by cytotoxic hypoxia, the excitatory amino acid l-glutamate or the N-methyl-2-phenylpyridinium ion 6, 20, 32, 34. In vivo, TGF-β1 reduces infarct size in experimental models of cerebral ischemia in both rabbits [14]and rats [25].

In AD TGF-β1 has been immunohistochemically localized to plaques located mainly in the molecular layer of the dentate gyrus, while in Down's syndrome, TGF-β1-positive plaques were preferentially located in the entorhinal cortex [38]. Furthermore, increased expression of TGF-β2 was found in large tangle-bearing neurons with widespread staining of glia in both non-dominantly inherited AD and in autosomal dominantly inherited AD [7]. The functions of the increased levels of TGF-β found in AD are unknown. This study was designed to determine if TGF-β 1, 2 or 3, can protect against neuronal degeneration caused by AβP fragment (25–35). Primary embryonic rat hippocampal neuronal cultures were treated with TGF-β 1, 2 or 3 before or after exposure to AβP and cell viability was measured. The results were confirmed in human and mouse neuroblastoma cells lines.

Section snippets

Cell culture

Primary rat hippocampal neuronal cultures were obtained from the brains of 16-day gestation fetal rats by dissection in ice-cold calcium- and magnesium-free Hank's balanced salts solution (HBSS) containing 100 μg/ml each streptomycin and penicillin (Gibco, USA). Dissected hippocampal tissue was collected in HBSS using a sterile pipette, placed in a 15 ml conical tube and centrifuged at 2000 rpm for 2 min. The resulting supernatant was discarded and replaced with 3 ml of 0.25% trypsin (Gibco,

Identification of primary rat hippocampal neuronal cultures with neuron-specific antibodies

Following treatment of primary cultures with uridine to inhibit astrocyte proliferation, the cells remaining in culture were identified as neurons by immunocytochemical staining with anti-NF and the A₂B₅ antibody. The A₂B₅ antibody stained the cell membrane and sprouting processes of cultured neurons (Fig. 1A,B). As shown in Fig. 1C, the neurons also showed cytoplasmic staining with the NF antibody. The majority of cells in the cultures did not stain with GFAP antibody, but astrocytes remaining

Discussion

This study demonstrates the ability of TGFs-β to protect against the cytotoxicity of AβP in a pure population of primary hippocampal neurons. Treatment of these neurons with either TGF-β 1, 2 or 3 for 24 h before addition of AβP for an additional 24 h, offered complete protection against the damaging effects of AβP. Hippocampal neurons pretreated in this manner showed MTT incorporation virtually equivalent to cells that had not been treated with AβP, while AβP treatment alone decreased MTT

Note added in proof

After submission of this manuscript, similar results demonstrating the ability of TGF-β to protect against AβP-induced neuronal damage were published [Prehn et al., Mol. Pharmacol., 49 (1996) 319].

Acknowledgements

We wish to thank Drs. Anita Roberts, Michael Sporn and Carol Lippa for helpful suggestions and critical reading of the manuscript.

References (41)

J Busciglio et al.
Methodological variables in the assesment of beta amyloid neurotoxicity
Neurobiol. Aging
(1992)
A Chalazonitis et al.
Transforming growth factor β has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor
Dev. Biol.
(1992)
K.C Flanders et al.
Effects of TGF-βs and bFGF in astroglial cell growth and gene expression in vitro
Mol. Cell. Neurosci.
(1993)
P.A Gruppuso et al.
Growth arrest induced by transforming growth factor β1 is accompanied by protein phosphatase activation in human keratinocytes
J. Biol. Chem.
(1991)
M.B Hansen et al.
Re-examination and further development of a precise and rapid dye method for measuring cell growth/ cell kill
J. Immunol. Methods
(1989)
N.D Klempt et al.
Hypoxia-ischemia induces transforming growth factor β1 mRNA in the infant rat brain
Mol. Brain Res.
(1992)
N.W Kowall et al.
In vivo neurotoxicity of beta-amyloid [β(1–40)] and the β(25–35) fragment
Neurobiol. Aging
(1992)
A Logan et al.
Enhanced expression of transforming growth factor β1 in the rat brain after a localized cerebral injury
Brain Res.
(1992)
J.C Martinou et al.
Transforming growth factor β1 is a potent survival factor for rat embryo motoneurons in culture
Dev. Brain Res.
(1990)
T.E Morgan et al.
TGF-β1 mRNA increases in macrophages/microglial cells of the hippocampus in response to deafferentation and kainic acid-induced neurodegeneration
Exp. Neurol.
(1993)

U Monning et al.

Transforming growth factor β mediates increase of mature transmembrane amyloid precursor protein in microglial cells

FASEB Lett.

(1994)

C.J Pike et al.

Cultured GABA-immunoreactive neurons are resistant to toxicity induced by β-amyloid

Neuroscience

(1993)

C.J Pike et al.

In vitro aging of β-amyloid protein causes peptide aggregation and neurotoxicity

Brain Res.

(1991)

D.K Rush et al.

Intracerebral β-amyloid (25–35) produces tissue damage: is it neurotoxic?

Neurobiol. Aging

(1992)

K Unsicker et al.

Transforming growth factor beta isoforms in the adult rat central and periperal nervous systems

Neuroscience

(1991)

J Waite et al.

Solvent effects on beta protein toxicity in vivo

Neurobiol. Aging

(1992)

J.H Bottenstein et al.

Growth of a rat neuroblastoma cell line in serum-free supplemented medium

Proc. Natl. Acad. Sci USA

(1979)

C.C Chao et al.

Transforming growth factor-β protects human neurons against β-amyloid-induced injury

Mol. Chem. Neuropathol.

(1994)

D Danielpour et al.

Sandwich enzyme-linked immunosorbent assay (SELISAs) quantitate and distinguish two forms of transforming growth factor (TGF-beta 1 and TGF-beta 2) in complex biological fluids

Growth Factors

(1989)

C.E Finch et al.

TGF-β1 is an organizer of responses to neurodegeneration

J. Cell. Biochem.

(1993)

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Research reportTransforming growth factors-β protect primary rat hippocampal neuronal cultures from degeneration induced by β-amyloid peptide

Abstract

Introduction

Section snippets

Cell culture

Identification of primary rat hippocampal neuronal cultures with neuron-specific antibodies

Discussion

Note added in proof

Acknowledgements

Neurobiol. Aging

Dev. Biol.

Mol. Cell. Neurosci.

J. Biol. Chem.

J. Immunol. Methods

Mol. Brain Res.

Neurobiol. Aging

Brain Res.

Dev. Brain Res.

Exp. Neurol.

FASEB Lett.

Neuroscience

Brain Res.

Neurobiol. Aging

Neuroscience

Neurobiol. Aging

Growth of a rat neuroblastoma cell line in serum-free supplemented medium

Proc. Natl. Acad. Sci USA

Transforming growth factor-β protects human neurons against β-amyloid-induced injury

Mol. Chem. Neuropathol.

Sandwich enzyme-linked immunosorbent assay (SELISAs) quantitate and distinguish two forms of transforming growth factor (TGF-beta 1 and TGF-beta 2) in complex biological fluids

Growth Factors

TGF-β1 is an organizer of responses to neurodegeneration

J. Cell. Biochem.

Research report
Transforming growth factors-β protect primary rat hippocampal neuronal cultures from degeneration induced by β-amyloid peptide