Elsevier

Brain Research

Volume 928, Issues 1–2, 22 February 2002, Pages 96-105
Brain Research

Research report
Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein

https://doi.org/10.1016/S0006-8993(01)03367-4Get rights and content

Abstract

The apolipoprotein E4 (apoE4) genotype is a major risk factor for Alzheimer’s disease (AD); however, the mechanism is unknown. We previously demonstrated that apoE isoforms differentially modulated neurite outgrowth in embryonic neurons and in neuronal cell lines. ApoE3 increased neurite outgrowth whereas apoE4 decreased outgrowth, suggesting that apoE4 may directly affect neurons in the brain. In the present study we examined the effects of apoE on neurite outgrowth from cultured adult mouse cortical neurons to examine if adult neurons respond the same way that embryonic cells do. The results from this study demonstrated that (1) cortical neurons derived from adult apoE-gene knockout (apoE KO) mice have significantly shorter neurites than neurons from adult wild-type (WT) mice; (2) incubation of cortical neurons from adult apoE KO mice with human apoE3 increased neurite outgrowth, whereas human apoE4 decreased outgrowth in a dose-dependent fashion; (3) the isoform specific effects were abolished by incubation of the neurons with either receptor associated protein (RAP) or lactoferrin, both of which block the interaction of apoE-containing lipoproteins with the low-density lipoprotein receptor-related protein (LRP). These data suggest a potential mechanism whereby apoE4 may play a role in regenerative failure and accelerate the development of AD.

Introduction

Apolipoprotein E (apoE), a 299-amino-acid protein, mediates the binding of lipid particles to lipoprotein receptors [6], [19]. In vivo, apoE probably functions in the distribution of lipids from an extracellular compartment for intracellular utilization [19]. ApoE-containing lipoproteins can bind to multiple members of the low-density lipoprotein receptor family [1], [31]. Humans have three major isoforms of apoE (apoE2, apoE3, and apoE4) that are produced by three alleles (ϵ2, ϵ3 and ϵ4) at a single gene locus on chromosome 19 [12], [32].

ApoE clearly has some important role in maintaining central nervous system (CNS) function [1], [31]. Inheritance of the ϵ4 allele of apoE increases the risk of Alzheimer’s disease (AD) whereas inheritance of the ϵ2 allele decreases the risk [9], [10], [26]. Numerous hypotheses have been proposed to explain apoE effects including formation and deposition of amyloid plaques and neurofibrillary tangles, antioxidant levels and the cholinergic system. However, the mechanism of apoE function is still unclear.

ApoE is synthesized and secreted primarily by astrocytes and microglia in the CNS [3], [24], [25]. ApoE is found associated with high-density lipoprotein-size particles in cerebrospinal fluid (CSF). Studies of dorsal root ganglion (DRG) neurons in vitro showed that apoE3 increased neurite outgrowth and apoE4 decreased outgrowth [22]. Similar observations of isoform-specific effects of apoE3 and apoE4 have been described in a murine neuroblastoma cell line (Neuro-2a) [2], [23]. Also, in a trkA transfected neuronal cell line (GT1) apoE3, but not apoE4, increased neurite extension [17]. Furthermore, embryonic hippocampal neurons from WT mice grown in the presence of astrocytes derived from apoE3 transgenic mice showed significantly greater neurite outgrowth than those grown in the presence of astrocytes from apoE4 transgenic mice [28]. Taken together, the data suggest that apoE isoforms can modify neurite outgrowth in embryonic neurons and in neuronal cell lines.

Previous studies exclusively used either embryonic cells or transformed cells to evaluate the effects of apoE isoforms. How apoE isoforms differentially alter neurite outgrowth remains unclear, as is the relevance of these previous findings to adult neurons and AD. We sought to answer these important questions by examining the effects of apoE isoforms on neurite outgrowth from cortical neurons derived from the brains of adult mice. We demonstrate that (1) adult mouse cortical neurons derived from apoE-deficient mice (apoE knockout, apoE KO) have significantly shorter neurites than neurons from normal mice; (2) human apoE3 enhances while human apoE4 inhibits neurite outgrowth in cortical neurons from adult apoE KO mice; and (3) the differential effects of apoE isoforms on neurite outgrowth in adult cortical neurons require the LRP.

Section snippets

Adult mouse cortical culture

Homozygous apoE KO mice (C57BL/6-Apoe<tm1Unc>) bred ten generations onto the C57BL/6 background and control mice (C57BL/6) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Cell culture medium, including Hibernate A, Neurobasal A, and B27 medium supplement were purchased from Life Technologies (Gaithersburg, MD, USA). Hibernate A is now available from BrainBits (Springfield, IL, USA). Glutamine and poly-d-lysine were purchased from Sigma (St. Louis, MO, USA). Papain was obtained from

Characterization of adult mouse cortical neuronal culture

We first modified previous techniques for the in vitro culture of neurons from adult mice [4]. The total recovery of viable cells after 4 days in culture averaged 65–70% of the cells plated. Fig. 1 shows results of immunofluorescent labeling of cell types in the culture. The majority of cells in the culture were neurofilament positive, representing ∼70% of the cells (Fig. 1A). GFAP positive cells comprised an average of 13% of total cells, and BSL1 stained cells represented about 14% of the

Discussion

The present study demonstrates that apoE modulates neurite outgrowth in adult neurons. ApoE KO mice had significantly diminished neurite outgrowth when compared to neurons in WT mice. Moreover, these data suggest that the decreased neurite outgrowth in apoE KO culture is not due to intrinsic properties of the neurons. Growth of apoE KO neurons in conditioned medium from WT mice was essentially identical to neurons from WT mice. Although the presence of apoE in the conditioned media is the most

Acknowledgements

Purified RAP was generously provided by Dr. Dudley Strickland (American Red Cross, Rockville, MD, USA). This work was supported by an Illinois Department of Public Health grant, and Eastern Illinois University CFR grants.

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    B.P. Nathan and Y. Jiang contributed equally to this study.

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