Amyloid precursor protein potentiates the neurotrophic activity of NGF

doi:10.1016/S0169-328X(97)00258-1

Molecular Brain Research

Volume 52, Issue 2, 15 December 1997, Pages 201-212

https://doi.org/10.1016/S0169-328X(97)00258-1 Get rights and content

Abstract

Cortical amyloid precursor protein (APP) is induced and secreted in response to subcortical lesions of cholinergic innervation. To understand the physiological role of the induced APP, we have characterized its neurotrophic activity on PC12 cells. Highly purified human APP₇₅₁ (50–1000 pM) induced outgrowth of neurites. The neurotrophic activity was inhibited by an antibody that was directed to the C-terminal portion of the secreted APP but not by an antibody directed to the KPI domain. The neurotrophic activity of APP was independent of the TrkA NGF receptor because neither phospholipase C-γ₁ nor TrkA exhibited tyrosine phosphorylations with APP treatment. Furthermore, APP stimulated neurite outgrowth from PC12 cells lacking TrkA receptors. At lower concentrations (10–50 pM), APP synergistically potentiated the neurotrophic effects of NGF when added with NGF or before NGF as a priming pretreatment. These results implicate APP, a rapidly induced protein in the injured cortex, as a potentiating agent that may render compromised neurons more responsive to low levels of NGF or other neurotrophins.

Introduction

Amyloid precursor protein (APP) is a family of integral membrane proteins that contain an extensive ectodomain which is secreted by proteolytic cleavage of the molecule at sites close to the plasma membrane. Disruptions of this secretory cleavage is postulated to be a pivotal step in the molecular pathology of Alzheimer's disease. However, the conservation of the primary sequence of the APP gene throughout phylogeny and its widespead and highly regulated expression all argue for an important normal physiological role of the protein in brain function. More specifically, the induction and secretion of cortical APP after subcortical lesions suggest that APP plays a role in the response of the cortex to loss of subcortical innervation 37, 38. Experiments using in vitro models showed that APP can act as a neuroprotectant 19, 30, mitogenic growth factor [29]or neurotrophic factor 1, 21, 27, 29. In vivo, administration of APP reduced nerve cell loss due to ischemia in rat brains [35]. Various forms of APP have been shown to induce neurites in vitro. Addition of soluble human APP to PC12 cells caused an elevation in the number of cells bearing small neurites [21]. A short peptide from the APP₆₉₅ sequence was found to promote neurite extension from B103 cells [15]. Growth of primary sympathetic chick ganglia or hippocampal neurons on culture dishes coated with purified human or bovine APP stimulated neurite outgrowth in a heparan sulfate proteoglycan-dependent manner [34]. Other studies have implicated APP in cellular adhesion [4]. Qiu et al. have found that APP present as an integral membrane protein on the cell surface induced neurites from primary rat hippocampal neurons [27]. These observations implicate APP as an injury-induced factor which may have growth factor properties.

The prototypic growth factor for neurons is nerve growth factor (NGF) which promotes the growth and survival of cholinergic neurons during development and regeneration. Consistent with this role is the observation that addition of exogenous NGF after lesioning of either the nucleus basalis of Meynert (nbM) or the fimbrial fornix significantly reduced the depletion of cortical cholinergic markers 6, 12, 13. Results from in vitro experiments suggest that APP and NGF may interact in their effects on neuronal cells. The expression and secretion of APP was elevated when PC12 cells were treated with NGF [28]. An antibody to APP was reported to inhibit the action of NGF in promoting neurite length, though not neurite number per cell, in PC12 cells [21]. Finally, NGF and APP appear to activate a common signal transduction pathway involving mitogen-activated protein kinase [9].

We hypothesized that the induced APP in the lesioned cortex interacts with NGF in response to the injury. To test this possibility, we have characterized APP neurotrophic activity to understand the interrelationship of APP and NGF in promoting neurite outgrowth in PC12 cells. We used purified APP at picomolar concentrations that were based upon our previous determinations of APP levels secreted into the CSF of lesioned animals [38]. APP was found to synergistically potentiate the neurotrophic activity of NGF.

Section snippets

Preparation of human APP₇₅₁

Human embryonic kidney 293 cells transfected with cDNA encoding the human sequence of APP₇₅₁ (PI6 cells) were used as the source of APP [24]. The secreted APP was purified essentially as described previously [24]. This form of APP is produced by α-secretase activity and therefore, contains a portion of the Aβ sequence at the carboxy-terminal of the secreted molecule. In brief, PI6 cells were grown on poly-d-lysine in DMEM/F12 media (1 : 1) containing 1 mM sodium pyruvate, 50 μM ethanolamine, and

Secreted form of APP₇₅₁ induced neurite outgrowth

Addition of highly purified APP (Fig. 1) to PC12 cells resulted in neurite outgrowth after 5–7 days (Fig. 2A). This activity was concluded to be specific because the neurotrophic activity was inhibited by an APP-specific antibody (8E5) (Fig. 3). An antibody directed specifically at the Kunitz protease inhibitor (KPI) domain of the APP (7H5, Athena Neurosciences) did not inhibit the neurotrophic activity even at 2 μg/ml concentrations (Fig. 3). Therefore, the neurotrophic activity was not likely

Discussion

Subcortical lesions of various neurotransmitter systems result in the in vivo induction and secretion of cortical APP 37, 38. Other insults to the brain that directly damage neurons also either increase levels of APP and/or it's related protein, APLP 3, 16, 25, 26, 31, 32, 33or alter APP processing [14]. These observations suggest a fundamental role for APP or APLP in brain injury response. In order to understand the physiological role of APP in the subcortically lesioned cortex, we have

Acknowledgements

We thank Dale Schenk, and Lisa McConologue of Athena Neurosciences for various anti-APP antibodies and PI6 cells, David Kaplan for nnr5 cells and assistance with TrkA characterization. Paul Ciesla provided excellent photographic expertise. Larisa Acevedo helped prepare graphic figures. We also acknowledge the constructive advice of Drs Moses Chao, Gordon Guroff, David Hawver, Nikki Holbrook, Don Ingram, Hynda Kleinman, John Kusiak, Derek LeRoith, and Laura Mamounas during the preparation of the

References (36)

A.J Dekker et al.
Effects of brain derived neurotrophic factor and nerve growth factor on remaining neurons in the lesioned nucleus basalis magnocellularis
Brain Res.
(1994)
M Goedert et al.
Nerve growth factor mRNA in peripheral and central rat tissues and in human central nervous system: lesion effects in the rat brain and levels in Alzheimer's disease
Mol. Brain Res.
(1986)
L Greene
A quantitative assay for nerve growth factor (NGF) activity employing a clonal pheochromocytoma cell line
Brain Res.
(1977)
L.A Greene et al.
The role of transcription-dependent priming in nerve growth factor promoted neurite outgrowth
Dev. Biol.
(1982)
V Haroutunian et al.
Attentuation of nucleus basalis of Meynert lesion-induced cholinergic deficits by nerve growth factor
Brain Res.
(1989)
T Kawarabayashi et al.
Expression of APP in the early stage of brain damage
Brain Res.
(1991)
S Korsching et al.
Cholinergic denervation of the rat hippocampus by fimbrial transection leads to a transient accumulation of nerve growth factor (NGF) without change in mRNA^NGF content
Neurosci. Lett.
(1986)
M.P Mattson et al.
Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β amyloid precursor protein
Neuron
(1993)
E.A Milward et al.
The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth
Neuron
(1992)
N Otsuka et al.
Rapid appearance of beta-amyloid precursor protein immunoreactivity in damaged axons and reactive glial cells in rat brain following needle stab injury
Brain Res.
(1991)

L.M Refolo et al.

Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC12 cell cultures

Biochem. Biophys. Res. Commun.

(1989)

T Saitoh et al.

Secreted form of amyloid β protein is involved in the growth regulation of fibroblasts

Cell

(1989)

D Schubert et al.

The expression of amyloid beta protein precursor protects nerve cells from β-amyloid and glutamate toxicity and alters their interaction with the extracellular matrix

Brain Res.

(1993)

R Siman et al.

Expression of β-amyloid precursor protein in reactive astrocytes following neuronal damage

Neuron

(1989)

H Araki et al.

Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture

Biochem. Biophys. Res. Commun.

(1991)

S.W Barger et al.

Role of cyclic GMP in the regulation of neuronal calcium and survival by secreted forms of beta-amyloid precursor

J. Neurochem.

(1995)

J.G Beeson et al.

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain

J. Comp. Neurol.

(1994)

K.C Breen et al.

Beta amyloid precursor protein mediates neuronal cell-cell and cell-surface adhesion

J. Neurosci. Res.

(1991)

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Research reportAmyloid precursor protein potentiates the neurotrophic activity of NGF

Abstract

Introduction

Section snippets

Preparation of human APP751

Secreted form of APP751 induced neurite outgrowth

Discussion

Acknowledgements

Brain Res.

Mol. Brain Res.

Brain Res.

Dev. Biol.

Brain Res.

Brain Res.

Neurosci. Lett.

Neuron

Neuron

Brain Res.

Biochem. Biophys. Res. Commun.

Cell

Brain Res.

Neuron

Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture

Biochem. Biophys. Res. Commun.

Role of cyclic GMP in the regulation of neuronal calcium and survival by secreted forms of beta-amyloid precursor

J. Neurochem.

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain

J. Comp. Neurol.

Beta amyloid precursor protein mediates neuronal cell-cell and cell-surface adhesion

J. Neurosci. Res.

Research report
Amyloid precursor protein potentiates the neurotrophic activity of NGF

Preparation of human APP₇₅₁

Secreted form of APP₇₅₁ induced neurite outgrowth