Elsevier

Neurobiology of Aging

Volume 36, Issue 10, October 2015, Pages 2737-2747
Neurobiology of Aging

Regular article
Herpes simplex virus type 2 infection induces AD-like neurodegeneration markers in human neuroblastoma cells

https://doi.org/10.1016/j.neurobiolaging.2015.06.014Get rights and content

Abstract

Herpes simplex virus (HSV) types 1 and 2 are neurotropic viruses that establish lifelong latent infections in neurons. Mounting evidence suggests that HSV-1 infection is involved in the pathogenesis of Alzheimer's disease (AD). The relationships between other herpesvirus infections and events associated with neurodegeneration have not, however, been extensively studied. The present work reports that HSV-2 infection leads to the strong accumulation of hyperphosphorylated tau and the amyloid-β peptides Aβ40 and Aβ42 (all major pathological hallmarks of AD) in human SK-N-MC neuroblastoma cells. Infection is also associated with a marked reduction in the amount of Aβ40 secreted and in the proteolytic fragments of the amyloid-β precursor protein (APP) (secreted APPα and the α-C-terminal fragment). These results indicate that HSV-2 infection inhibits the nonamyloidogenic pathway of APP processing and impairs Aβ secretion in these cells. In addition, HSV-2 induces the accumulation of intracellular autophagic compartments containing Aβ due to a failure in the late stages of autophagy. To our knowledge, this is the first report to show that HSV-2 infection strongly alters the tau phosphorylation state, APP processing, and autophagic process in human neuroblastoma cells, leading to the appearance of AD-like neurodegeneration markers.

Introduction

Alzheimer's disease (AD), the single most common cause of dementia, is characterized by massive neuronal damage leading to cerebral atrophy and the loss of cognitive function. The major neuropathological lesions of AD are brain β-amyloidosis and neurofibrillary degeneration; the former is characterized by plaques of extracellular amyloid-β peptide (Aβ) in the brain parenchyma and blood vessels, while the second involves the intracellular accumulation and aggregation of abnormally hyperphosphorylated tau protein (a microtubule-associated protein) in the form of neurofibrillary tangles (Braak and Braak, 1992). Two distinct forms of AD are recognized: familial and sporadic. Familial AD is caused by mutations in the genes for amyloid-β precursor protein (APP), presenilin-1, and presenilin-2. However, most cases of AD (∼99%) are of the sporadic type and usually involve people >65 years (Tanzi, 2012).

Sporadic AD is a highly complex disease for which neither the causal agent(s) nor the molecular mechanisms behind its pathogenesis are well known. A growing body of literature suggests, however, that cerebral infections may be risk factors for neurodegenerative disease, and certainly numerous studies have revealed an association between herpes simplex virus type 1 (HSV-1) and AD (Itzhaki, 2014, Piacentini et al., 2014). For example, HSV-1 DNA is present in the brains of a high proportion of people with AD (Jamieson et al., 1991), mainly within amyloid plaques (Wozniak et al., 2009b), the virus has been shown capable of inducing the main hallmarks of AD pathology (Alvarez et al., 2012, Santana et al., 2012b, Wozniak et al., 2007, Wozniak et al., 2009a), and the analysis of data gathered in genome-wide association studies involving thousands of AD patients and controls identified a set of AD-linked gene variants that might increase brain susceptibility to viral infections, particularly HSV-1 infection (Porcellini et al., 2010). Few studies, however, have focused on the role of other herpesviruses in AD, even though 5—herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), human herpes virus 6, varicella-zoster virus and Epstein-Barr virus (EBV)—have been detected in the brains of elderly individuals and AD patients (Carbone et al., 2014, Hemling et al., 2003, Lin et al., 2002).

Like HSV-1 (which causes cold sores), HSV-2 (which causes genital herpes) belongs to the alpha-herpesvirus subfamily. These viruses can reach the sensory neurons that innervate the site of primary infection, and establish lifelong latent infections. Reactivation from the latent state causes recurrent disease, which is particularly common in immunocompromised hosts (Steiner and Benninger, 2013). A recent study estimates 57% of US adults to be infected with HSV-1 and 17% with HSV-2 (Koelle and Corey, 2008). It is well known that HSV-2 can infect the brain and cause neurological problems such as encephalitis and meningitis in neonates and encephalitis in adults (Berger and Houff, 2008). HSV-2 DNA, however, is found in the brains of far fewer people than HSV-1 DNA (both in those with AD and age-matched controls) (Lin et al., 2002). There are few comparative studies of HSV-1 and HSV-2 central nervous system infections, but it has traditionally been thought that HSV-2 causes fewer cases of herpes simplex encephalitis in adults than HSV-1 (∼10% compared to ∼90%) (Berger and Houff, 2008). However, a recent report suggests that HSV-1 and HSV-2 account for similar numbers (Moon et al., 2014). These discordant findings may result from ethnic or regional differences between the study populations. In addition, the latter study has some limitations—all patients belong to a single center and it is a retrospective study with not well-defined criteria for categorization of neurological diseases. Further studies with well-designed prospective studies in multiple centers and regions are required to confirm that HSV-2 is a major cause of severe encephalitis in adults.

Our group and others have reported HSV-1 infection to modify tau phosphorylation (Alvarez et al., 2012, Wozniak et al., 2009a, Zambrano et al., 2008), APP proteolytic processing (De Chiara et al., 2010, Santana et al., 2012b, Wozniak et al., 2007), and autophagy (Gobeil and Leib, 2012, Santana et al., 2012a), all of which have been associated with the pathogenesis of AD. However, the effect of HSV-2 infection on these processes has not been extensively studied. The present work reveals that, like HSV-1 infection, HSV-2 infection can provoke the appearance of the neurodegeneration markers characteristic of AD in different cell models.

Section snippets

Drugs, plasmids, and antibodies

Heparin (10 μg/mL), rapamycin (0.2 μg/mL), and bafilomycin A1 (100 nM) were purchased from Sigma. Leupeptin (100 μM) was supplied by Roche. 4′, 6-diamidino-2-phenylindole (DAPI; 5 μg/mL) and ammonium chloride (NH4Cl; 20 mM) were obtained from Merck.

The GFP–light chain 3 (LC3) expression vector (pGFP-LC3) and the mCherry-GFP-LC3 construct (dtLC3) were kind gifts from T. Yoshimori and N. Mizushima (Kabeya et al., 2000), and T. Johansen (Pankiv et al., 2007), respectively. Rabbit anti-HSV

HSV-2 induces the accumulation of hyperphosphorylated tau

Before all else, the HSV-2 infection parameters for the SK-N-MC cells were optimized. In this cell model, HSV-2 establishes a lytic replication cycle resulting in cell lysis ∼20–22 hours after infection. In this work, all infections were performed at a moi of 10 pfu/cell because this viral dose guarantees that almost all cells become infected, as revealed by confocal images of HSV-2-infected cells, visualized via the use of an antibody that recognizes glycoproteins B and D of the virus (

Discussion

AD is a multifactorial disorder that apparently involves different etiopathogenic mechanisms. One of the factors involved may be infectious agents that are able to colonize the brain and thus escape the immune response. Infection by different members of the herpesvirus family may be a risk factor for AD. These viruses are highly prevalent in the human population and the infection rate increases with age, they can infect neurons, and they establish lifelong latent infections that cannot be

Disclosure statement

Soraya Santana is currently employed by the commercial company BioPharma Division, Neuron Bio, Granada, Spain. The other authors declare no actual or potential conflicts of interest.

Acknowledgements

This work was supported by grants from the Ministerio de Economía y Competitividad (SAF2010-15558; URL: http://www.mineco.gob.es/portal/site/mineco) and Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas CIBERNED (PI2010/09-8; URL: http://www.ciberned.es). The authors thank Drs. T. Yoshimori and N. Mizushima for providing the GFP-LC3 expression vector; Dr. T. Johansen for providing the mCherry-GFP-LC3 construct and Dr. E. Tabares for providing the rabbit anti-HSV-1

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    1

    These authors contributed equally to this work.

    2

    Present address: BioPharma Division, Neuron Bio, Parque Tecnologico de Ciencias de la Salud, Edificio BIC, Avda. de la Innovación 1, 18,100 Armilla, Granada, Spain.

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