Elsevier

Neurobiology of Aging

Volume 29, Issue 1, January 2008, Pages 71-77
Neurobiology of Aging

Isoform-specific effects of ApoE on HSV immediate early gene expression and establishment of latency

https://doi.org/10.1016/j.neurobiolaging.2006.09.006Get rights and content

Abstract

Alzheimer's disease (AD) is a common and devastating neurodegenerative disease in which most cases are of unknown, sporadic origin. In addition to age, the most prevalent known risk factor for developing AD is carriage of the ɛ4 allele of Apolipoprotein E (ApoE). Carriage of the ɛ2 or ɛ3 allele of ApoE confers protection or no change in risk for AD, respectively. Latent herpes simplex virus type 1 (HSV-1) infection in the brain concurrent with ApoE4 carriage exacerbates risk for AD, suggesting that these two factors interact to promote neuronal dysfunction and degeneration in selective brain areas. Indeed, HSV-1 DNA has been found in regions primarily affected by AD, such as the temporal lobes, hippocampus, and neocortex. We hypothesize that HSV-1 infection in the background of ApoE4, but not ApoE2 or ApoE3, promotes an environment more conducive to neuronal degeneration. To investigate this idea, we have utilized transgenic mice that express human ApoE2, 3, or 4 alleles from astrocytes in a murine ApoE −/− background. We find that carriage of the different ApoE alleles dramatically affects HSV-1 immediate early gene expression as well as the establishment of latency. Both of these factors are poised to impact neuronal viability, inflammation, and viral spread. Our data support the concept that HSV-1 and ApoE4 interact to provide an environment conducive to the development and/or spread of AD.

Introduction

Apolipoprotein E (ApoE) is a brain-enriched component of very-low-density lipoprotein (VLDL) whose functional roles extend beyond the traditional one of cholesterol transport and receptor mediated uptake of lipoprotein particles into cells. The uptake of esterified cholesterol mediated by ApoE and the LDL receptor-related protein (LRP) into neurons in the aged brain is thought to precede synaptic terminal repair and/or synthesis [27]. ApoE also appears to be involved with the delivery of lipids to axons in the setting of injury and regeneration [5]. Many of the brain-specific functions of ApoE are differentially manifest depending on the isoform of ApoE present (reviewed in [20]). There are three human ApoE alleles, found at the same gene locus, designated ɛ2, ɛ3, ɛ4, which differ in the amino acids found at positions 112 and 158. These different alleles encode proteins ApoE2, E3, and E4 that have varying actions with respect to the novel brain roles of ApoE. ApoE is produced by astrocytes, but is readily taken up into neurons, where it can be localized in immunostained sections of normal brain [21], [23]. In contrast, Alzheimer disease (AD) brains show ApoE localized to extracellular senile plaques. Indeed, ApoE and the neurotoxic peptide amyloid-β (Aβ) form insoluble complexes in vitro[21], [23], [33]. The landmark observations that carriage of the Apoɛ4 allele correlates with increased brain deposition of Aβ and, even more striking, early onset AD, sparked renewed interest in the role of ApoE in both normal and diseased brains [4], [28], [30], [31]. In fact, fibrillar ApoE-Aβ structures form more rapidly and are denser when ApoE4 is used instead of E3 [29], [34]. ApoE4 is also associated with reduced neuronal remodeling [1], lower density of cholinergic neurons in AD brains, reduced choline acetyltransferase activity, and clinical response to tacrine (a cholinesterase inhibitor) [27], as well as lack of neurite branching and outgrowth [6], [8], [24], [35] and enhanced depolymerization of microtubules [25]. All of these E4 specific effects mirror what is seen in the AD brain, and may help to explain why carriage of an ɛ4 allele hastens the onset and severity of AD.

Although ApoE4 is a risk factor for AD, it alone appears insufficient to cause disease. One intriguing possibility for how ApoE may act to influence disease onset and progression was posited to reflect interaction with a human pathogen. It was suggested that a combination of ɛ4 allele and subclinical herpes simplex virus 1 (HSV1) infection of the brain synergistically predispose one to AD [10], [12]. Other findings support this assertion. People with cold sores harboring reactivated HSV1 are significantly more likely to carry an ɛ4 allele than those with HSV1 infection that do not experience cold sores [11]. Purified glycoprotein B (gB) from HSV1 interacts directly with ApoE [9], and both ApoE and HSV1 can enter cells via a heparan sulfate glycoprotein (HSPG) receptor, resulting in their accumulation inside the cell [15]. Recently, it was demonstrated that ApoE is required for HSV1 spread to the brain via the hematogenous route [3]. In that study, real time quantitative PCR (QPCR) was used to demonstrate viral DNA present in different tissues and brain regions of control mice and mice lacking the ApoE gene (hemi- or homozygous knockout mice). Knockout mice (KO) had significantly less HSV DNA in the central nervous system than controls, with the most marked decreases found in midbrain and cerebellum [3]. Examination of total brain and the midbrain region revealed that mice hemizygous at the APOE locus had HSV1 DNA levels in between homozygous knockouts and controls, indicating a gene dosage effect [3]. To further explore the possibility that ApoE, and particularly E4, can regulate HSV1 gene expression or spread in the brain, we have utilized isoform-specific transgenic mice that express human E2, E3, E4, or no ApoE in astrocytes from the glial fibrillary acidic protein (GFAP) promoter [32]. We find that ApoE4 and ApoE KO mice support considerably more HSV1 immediate early (IE) gene expression in neurons, but far less expression of the latency associated transcript in sensory ganglia. The implications for an ApoE-HSV1 interaction in the etiology of AD are discussed.

Section snippets

Mice

ApoE transgenic mice were received from Dr. Jonathan Smith, Rockefeller University, New York, NY [32]. Briefly, C57BL/6 mice expressed one copy of human ApoE2, 3, or 4 from the GFAP promoter on a mouse ApoE deficient background. KO mice that did not express murine or human ApoE were also used in the study. The genotypes of all parent mice were confirmed by PCR of tail DNA prior to their being used in a cross. Mice were fed ad libitum and housed at 5 animals or fewer per cage. All procedures

Results

Our studies aim to investigate whether ApoE4 and HSV-1 infection may conspire to promote neurodegeneration. One assumption is that the three isoforms of ApoE interact with HSV-1 differently. To determine whether ApoE isoform specific differences in IE gene expression or viral entry into cells exist, primary neuronal cortical cultures derived from E14.5 ApoE2, E3, E4 or KO embryos were infected with helper-virus free HSVlac. This amplicon vector relies on the HSV IE 4/5 promoter to drive

Discussion

The principle finding in this study using isoform-specific transgenic ApoE mice and ApoE KO mice is that the mice lacking ApoE or expressing the human E4 allele provide a more conducive environment for HSV replication and spread compared to mice expressing E2 or E3. These ApoE isoform-specific differences did not affect overall mortality of HSV-1 infected mice, suggesting that the effects we observe may be limited to the nervous system. KO and E4 mice supported higher IE gene expression from

Acknowledgements

We thank L. Salamone and L. Prifti for animal husbandry and surgical assistance. We thank D. Lioy and J. Stoklosa for technical assistance with surgical and PCR procedures. We thank Dr. W. Bowers, R. Giuliano, and D. Howard for assistance with virus preparation. We received ApoE transgenic mice from Dr. J. Smith, of Rockefeller University. We received HSV McKrae strain from Dr. J. Pepose, of Washington University, St. Louis. We thank Dr. D. Leib for helpful discussions regarding McKrae strain

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