Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model

Abstract

Senile amyloid plaques are one of the main hallmarks of Alzheimer’s disease (AD). They correspond to insoluble deposits of amyloid-β peptides (Aβ) and are responsible for the inflammatory response and neurodegeneration that lead to loss of memory. Recent data suggest that Aβ possess antimicrobial and anti-viral activity in vitro. Here, we have used cocultures of neuroglioma (H4) and glioblastoma (U118-MG) cells as a minimal in vitro model to investigate whether Aβ is produced by neuroglioma cells and whether this could result in protective anti-viral activity against HSV-1 infection. Results showed that H4 cells secreted Aβ₄₂ in response to HSV-1 challenge and that U118-MG cells could rapidly internalize Aβ₄₂. Production of pro-inflammatory cytokines TNFα and IL-1β by H4 and U118-MG cells occurred under basal conditions but infection of the cells with HSV-1 did not significantly upregulate production. Both cell lines produced low levels of IFNα. However, extraneous Aβ₄₂ induced strong production of these cytokines. A combination of Aβ₄₂ and HSV-1 induced production of pro-inflammatory cytokines TNFα and IL-1β, and IFNα in the cell lines. The reported anti-viral protection of Aβ₄₂ was revealed in transfer experiments involving conditioned medium (CM) of HSV-1-infected H4 cells. CM conferred Aβ-dependent protection against HSV-1 replication in de novo cultures of H4 cells challenged with HSV-1. Type 1 interferons did not play a role in these assays. Our data established that H4 neuroglioma cells produced Aβ₄₂ in response to HSV-1 infection thus inhibiting secondary replication. This mechanism may play a role in the etiology of AD.

Keywords

Alzheimer’s disease amyloid-β peptides cocultures glial cells herpes simplex virus-1 (HSV-1)neuronal cells viral replication inhibition

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by irreversible destruction of specific regions of the brain. AD is clinically identified by initial progressive loss of memory, impaired language skills, and ongoing manifestations of cognitive functional alterations, such as judgment and decision making, individual self-autonomy and, in a large number of cases, uncontrolled behavioral disturbances [1, 2]. Currently, AD is the most common form of dementia worldwide [3]. It is estimated that nearly 35.6 million people currently suffer from this form of dementia and it is expected that this number will triple in the next 40 years or so [4] largely due to increased life expectancy of the global population, as aging is the most important risk factor of AD [5].

There are two clinical forms of AD. The early onset of AD (EOAD) corresponds to the genetic and familial form whereas the late onset of AD (LOAD) is the sporadic manifestation of the disease that generally occurs after the age of 65. However, both forms are characterized by similar pathological changes namely, irreversible neuronal loss, deposits of cortical senile plaques and neurofibrillary tangles in the brain of affected patients [6, 7]. The hallmark of LOAD is the intracellular accumulation of tangles of hyperphosphorylated tau protein and the extracellular deposits (senile plaques) of short amyloid- β peptides (Aβ) 1–40 (Aβ₄₀) and 1–42 (Aβ₄₂) derived from successive cleavage of the amyloid-β precursor protein (AβPP), through the amyloidogenic pathway. The amyloidogenic pathway is initiated by the action of β-secretases (BACE-1) that release the ectodomain of AβPP, which becomes the substrate of the γ-secretase complex that generates the Aβ fragments [8 –10] that are secreted [11]. Accumulation of deposits of Aβ in a filamentous (insoluble) form is associated with neuronal degeneration and cortical atrophy [2, 12]. The exact cause of LOAD remains elusive but has been proposed to be linked to a host of factors such as gene susceptibility [13, 14], metabolic disorders [15, 16], brain inflammation [17 –19], and brain infection by several types of pathogens [20 –22]. In addition, environmental factors, family history, and vascular risk factors such as hypertension and hypercholesterolemia have been suggested as additional factors that may contribute to AD [23, 24]. The mechanism of Aβ accumulation in LOAD is not fully understood but appears to be caused by overproduction or by a defect in clearance and degradation by microglial cells, or both [25]. In addition, Aβ clearance is altered by age-related immune changes (immunosenescence and inflamm-aging) [26 –29].

Chronic inflammation of the brain (neuroinflammation) and the peripheral immune system is a fundamental characteristic of AD [30 –33]. Neuroinflammation is triggered by microglia and astrocyte activation and, infected neurons in some cases. In this connection, microglia and astrocytes have been shown to proceed from a resting to an activated inflammatory state following brain infection or insult [30, 34]. Upregulation of microglia and astrocyte activity results in production of pro-inflammatory cytokines, such as TNFα, IL-1β, and IL-6, and reactive oxygen species (ROS) and nitric oxide (NO) [35]. These mediators are responsible for apoptosis or necrosis of damaged neurons [35]. In addition, microglia respond to Aβ aggregates and plaques through a number of receptors, namely RAGE, scavenger receptors, CD36, Fc receptors, and TLR [36]. Occupation of these receptors induces the switch to a neurotoxic state that sustains neuroinflammation and induces Aβ phagocytosis and degradation. Although the amyloid hypothesis is a signature of LOAD [37], its role as a cause of AD is still much debated, especially in view of the failure of Aβ-targeted immunotherapies [38 –42].

Viral infections have been suggested to be a contributing factor to AD [20 –22]. For instance, herpes simplex virus type 1 (HSV-1) is frequently found in AD patients’ brain [22, 43] where it colocalizes within amyloid plaques [44]. In addition, HSV-1 sero-positive individuals are at increased risk of AD [45]. Other members of the herpes virus family, namely HSV-2, CMV, and HHV-6, have also been detected in the brain of AD patients [46] or have been associated with its pathogenesis [43]. HSV-1 is a virus that infects approximately 90% of the human population early (before 10 years of age) and is able to remain latent during the whole life of the infected individuals [47]. The exact percentage of individuals with HSV-1 in the brain is still uncertain but in any case is increasing with age and has been estimated by different studies to be around 70% [48, 49]. HSV-1 escapes immune recognition by remaining latent in trigeminal ganglions but can be activated under conditions of immunodeficiency or stress. Under these conditions, HSV-1 re-infects the host [50] and colonizes the hippocampus and fronto-temporal lobes [51]. In cases of strong immunodeficiency, re-activation of HSV-1 can trigger lethal herpetic encephalopathy, which also occurs in the same areas of the brain as those affected in AD (hippocampus and frontal and temporal cortical lobes) [52 –54].

A new picture is slowly emerging with respect to a protective role of Aβ against pathogenic agents. For instance, we [55] and one other group [56] have presented evidence that Aβ possessed antiviral activity against enveloped HSV-1 as well as H3N2 and H1N1 influenza A virus strains, respectively, in in vitro assays. In addition, Aβ have been reported to display antimicrobial activity against a host of clinically relevant bacteria and yeast [57]. The bulk of these observations suggests that Aβ may belong to a novel class of antimicrobial peptides that protect against neurotropic enveloped virus infections such as HSV-1 and influenza virus and against other neuroinfections [21]. Here, we sought to obtain further evidence for Aβ-dependent protection against HSV-1 infection using an in vitro model of coculture of neuroglioma (H4) and glioblastoma (U118-MG) cell lines. Results showed that HSV-1 triggered a time-dependent Aβ₄₂ production by H4 cells but the absence of Aβ₄₀ production. U118-MG cells produced low amounts of Aβ₄₂ or Aβ₄₀. Production of Aβ was also low in cocultures of H4/U118-MG cells exposed to HSV-1, due to rapid uptake of Aβ by U118-MG cells. Together, Aβ₄₂ and HSV-1 induced production of pro-inflammatory cytokines TNFα and IL-1β, and IFNα in the cell lines. Culture medium of H4 cells exposed to HSV-1 (defined as conditioned medium or CM) conferred protection against infection of de novo cultures of H4 cells by HSV-1 due to the presence of Aβ in the CM. Type I interferons (IFNα, IFNβ) were not responsible for the protective effect. Taken together, our data further support our previous observations [55] that Aβ display anti-viral activity against HSV-1 infection in in vitro assays and extend these observations to demonstrate that Aβ is induced by HSV-1 infection in an in vitro model of coculture of neuroglioma andglioblastoma cells.

MATERIALS AND METHODS

Cell cultures and cocultures

H4 (ATCC HTB-148), a human neuroglioma cell line, and U118-MG (ATCC HTB-15), a human glioblastoma cell line, came from ATCC (American Type Culture Collection, Manassas, VA). MRC-5, a human lung fibroblast cell line, was obtained from Diagnostic Hybrids (Athens, OH). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin G (2.5 I.U./ml), and streptomycin (50 μg/ml). Serum, medium, and antibiotics were from Wisent Bioproducts (St-Bruno, QC). Individual cell cultures were allowed to reach confluence before use. In the case of cocultures of H4 and U118-MG, each cell line was grown to approximately 70% confluence. H4 cells were detached by trypsin treatment and added to adherent U118-MG cells in fresh medium. Cocultures were left to grow for 24 h before use.

Synthetic amyloid-β peptides, antibodies, and BACE-1 inhibitor

Synthetic Aβ₄₀, Aβ₄₂, and Alexa Fluor 555-conjugated Aβ₄₂ were obtained from Anaspec (Fremont, CA). Aβ were used at a concentration of 20 μg/ml for cytokine quantification experiments and at 5 μg/ml for phagocytosis experiment of Alexa Fluor 555-labeled Aβ₄₂ by U118-MG cells. These concentrations of Aβ peptides are in agreement with those used by other investigators [58 –61]. Furthermore, we also tested concentrations of 1 μg/ml and 10 μg/ml. Best and most consistent results were obtained at the above concentrations [55]. Amino acid sequences of synthetic peptides used in this study have been reported [55]. A neutralizing anti-human α-IFN monoclonal antibody was purchased from EMD Millipore (EMD Millipore, Etobicoke, ON) and used at a 100 ng/ml concentration. Anti-human β-interferon neutralizing polyclonal antibodies (EMD Millipore, Etobicoke, ON) were used at a concentration of 10 units/ml. Aβ-secretase (BACE-1) inhibitor was obtained from EMD Millipore (product #565749) and used at a 2.5 μM concentration.

HSV-1

HSV-1 was an isolate obtained from the clinical microbiology and virology laboratory of the Centre Hospitalier Universitaire de Sherbrooke (CHUS)[55].

Quantification of Aβ by ELISA

A sandwich ELISA method was used to determine Aβ₄₀ and Aβ₄₂ concentrations in cell supernatants. ELISA-designed 96-well plates were coated(5 μg/ml) with N-terminal anti-Aβ antibody (Rockland, Limerick, PA) in coating buffer (NaHCO₃, 100 mM, Na₂CO₃, 34.9 mM, pH 9.5) and incubated at 4°C overnight. After 3 washes with PBS-T (PBS containing 0.05% Tween-20), ELISA plates were subjected to a blocking step using a blocking buffer (PBS containing 10% FBS, 200 μl/well) for 1 h at roomtemperature. Three additional washes were performed with PBS-T buffer. A calibration curve was constructed by adding serial dilutions of Aβ₄₀ or Aβ₄₂ over a range of concentrations from 3.91 pg/ml to 10 μg/ml in coating buffer. Incubations were done at room temperature for 3 h, followed by 3 washes with PBS-T. Quantification of Aβ in cell supernatants was performed in the same way. Primary antibody against Aβ₄₀ or Aβ₄₂ (Anaspec) was diluted in coating buffer at 1 μg/ml and added to the plates for 1 h at room temperature. After 3 washes (PBS-T), HRP-conjugated anti-mouse IgG (Abcam, Cambridge, MA) was added to each well at 1:500 final dilution in coating buffer, incubated for 30 min at room temperature and then washed 5 times (PBS-T). TMB substrate (BioLegend, San Diego, CA) was mixed and 100 μl were added to each well. The colorimetric reaction was stopped by addition of 1N H₂SO₄ (100 μl). Plates were read at 450 nm using a micro plate reader (Viktor X5, Perkin Elmer, Waltham, MA). Concentrations were determined by reference to the calibration curve, after subtraction of the blank values.

Measure of BACE-1 activity

SensoLyte 520 β-secretase Assay Kit “Fluorimetric” (Anaspec) was used to quantitate the activity of BACE-1 in H4 cells lysates following HSV-1 infection by measuring the fluorescent signal (Ex/Em = 485/535 nm) of the fluorescent peptide substrate provided in the kit. Activity was calculated by comparison with an internal control and is represented as relative fluorescent units (RFU).

Cytokine quantification

Multiplex immunobead assay technology (Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, EMD Millipore Corp., Billerica, MA) was used to simultaneously detect three human cytokines (TNFα, IL-1β and IFNα) in small samples of cell supernatants. Cytokines in cell culture supernatants were quantified at baseline without cell treatment and 16 h later after addition of Aβ₄₀ or Aβ₄₂ in the absence or presence of HSV-1 challenge. Supernatants were processed according to the manufacturer’s recommendations (thawing and centrifugation) and quantification was performed using the Multiplex Assay Analysis Software (EMD Millipore). Analyte concentrations were calculated based on respective standard curves for each analyte.

DNA isolation and amplification of cells treated with Aβ and HSV-1

MRC-5, H4, and U118-MG cells were cultured in DMEM (100 μl) in 96-well plates and then treated under various conditions. HSV-1 was titrated by end point dilution with MRC-5 cells using serial dilutions of the virus in 96-well plates. Based on this titration, cells were infected in all experiments at a ratio of 0.01 ID₅₀ per cell. Cells were treated with BACE-1 inhibitor or neutralizing antibodies and exposed to HSV-1, as described in the Figure legends. In most cases, treatments consisted of addition of BACE-1 inhibitor and/or antibodies for 2 h to cell cultures, followed by exposure to HSV-1. Assays designed to test the protective effect of Aβ were prepared as follows. Culture medium from H4 cell cultures not exposed to HSV-1 was added to de novo H4 cell cultures 2 h prior to addition of HSV-1. In another set of experiments, culture medium of HSV-1-infected H4 cells was harvested after 24 h and designated as conditioned medium (CM). The CM was added to de novo H4 cell cultures 2 h prior to addition of HSV-1. In each case, viral DNA replication was stopped by freezing the cell suspension 24 h post viral exposure. DNA was extracted by alkaline lysis and ethanol precipitation [62]. Aliquots (1 μl) were amplified by real-time PCR without purification to detect viral and β-actin DNA, as described [55]. Amplified viral DNA was normalized to amplified β-actin DNA (2exp(β-actin _Cq – viral _Cq) × 1000) to correlate viral DNA variations to cell number.

Analysis of Alexa Fluor 555-labeled Aβ₄₂ uptake by U118-MG cells using fluorescence microscopy

Alexa Fluor 555-labeled Aβ₄₂ was added (5 μg/ml) to confluent U118-MG cells. Cells were mounted using the ProLong Antifade kit (Life Technologies Inc. Burlington, ON) that contained DAPI DNA-stain. Images were recorded at room temperature using a Nikon Eclipse TE2000-S fluorescence microscope (Melville, NY) equipped with a CCD camera (Qimaging 32-0116A-122 Retiga 1300R Fast Cooled Mono 12 bit, Surrey, BC, Canada). Data were processed using Simple PCI software.

Cell viability

Cell viability was determined using the Trypan blue exclusion method.

Statistical analysis

Data were analyzed using the Graph-Pad Prism 5 (La Jolla, CA) or Excel software (Microsoft Canada Inc., Mississauga, ON). Statistical analyses were performed by two-tailed Student’s t-test.

RESULTS

Time- and β-secretase-dependent production of Aβ in H4 neuroglioma cells and U118-MG glioblastoma cells in response to HSV-1 infection

In a first series of experiments, we quantitated Aβ production by H4 and U118-MG cells in response to HSV-1 infection. Results showed that baseline production of Aβ₄₂ by H4 cells after 24 h in culture was low, amounting to 13.2 ± 3.9 pg/ml (Fig. 1A). In marked contrast, HSV-1 induced a significant (p < 0.05) 4-fold increase in production of Aβ₄₂ (52.2 ± 17.1 pg/ml) 24 h after infection of these cells (Fig. 1A). However, HSV-1 did not induce Aβ₄₀ production, under the same experimental conditions (Fig. 1A). Kinetic experiments revealed a progressive production of Aβ₄₂ following HSV-1 infection but there was no significant increased production of Aβ₄₀ at any time (Fig. 1B). Evidence that Aβ₄₂ production by the H4 neuroglioma cell line involved the established Aβ pathway was obtained by measuring BACE-1 activity. Results showed that basal activity in H4 cells infected with HSV-1 was similar at 2 h (886.3 ± 20.3 RFU) and 6 h (855.3 ± 20.3 RFU) but significantly increased (p < 0.0001) at 24 h (1193 ± 56 RFU) (Fig. 1C). HSV-1 challenge of U118-MG cells did not stimulate increased production of Aβ₄₂ which was 13.2 ± 5.1 pg/ml under basal conditions and 9.0 ± 3.2 pg/ml following challenge with HSV-1 for 24 h (Fig. 1D). Aβ₄₀ production was low (0.35 ± 0.25 pg/ml) in U118-MG cells under basal conditions but exposing the cells to HSV-1 for 24 h induced a significant (p < 0.01) but small (1.75 ± 0.26 pg/ml) increase in Aβ₄₀ production (Fig. 1D).

Effect of β-secretase inhibition on HSV-1 replication and Aβ₄₂ production in long-term cultures of H4 cells

We next investigated whether H4 cells were able to sustain replication of HSV-1 and continued productionof Aβ₄₂ in the absence or presence of BACE-1 inhibitor, over a 5-day period of time. Results showed that HSV-1 replication in the presence of BACE-1 inhibitor increased as a function of time (Fig. 2A). In contrast, HSV-1 replication was severely inhibited in cultures not containing BACE-1 inhibitor (Fig. 2A). These observations suggested that inhibition of HSV-1 replication was related to the presence of Aβ, in agreement with previous observations from our laboratory [55]. Further support for this interpretation was obtained by quantification of Aβ production in the absence of BACE-1 inhibitor. Results showed that HSV-1 induced sustained production of Aβ₄₂ over time up to 30 pg/ml (Fig. 2B). As expected, the presence of BACE-1 inhibitor efficiently blocked Aβ production (Fig. 2B). H4 cells remained viable (93.8 ± 1.9% ) over the 5-day period of culture and the continued presence of HSV-1 did not significantly affect cell viability (80.2 ± 12.5% ) (Fig. 2C). The presence of BACE-1 inhibitor decreased cell viability over the 5-day period of culture, although cells remained viable to the level of 59.0 ± 25.5% . Exogenous addition of Aβ₄₂ reduced cell viability to 46.4 ± 12.5% over the same period of time (Fig. 2C), an observation which is likely due to the reported cytotoxicity of Aβ₄₂ [63].

Aβ production in cocultures of H4 and U118-MG cells in response to HSV-1 infection

The major objective of the current investigation was to use an in vitro model of neuronal/glial cell interactions to further investigate the role of Aβ in protection against HSV-1 infection. Cocultures of H4 and U118-MG cells were set and Aβ production quantitated. Results showed absence of Aβ₄₀ accumulation under basal conditions or when the cells had been challenged with HSV-1 for 24 h (Fig. 3A). These data suggested that U118-MG cells did not influence the previously observed low levels of Aβ₄₀ production by H4 cells. Unexpectedly, data showed that Aβ₄₂ concentrations were slightly lower in cocultures, with values of 7.7 ± 3.1 pg/ml under basal conditions, as opposed to 13.3 ± 3.9 pg/ml in H4 cultures alone. A similar observation was made under conditions of HSV-1 infection for 24 h. In this case, values were 11.2 ± 3.6 pg/ml in cocultures as opposed to 52.2 ± 17.1 pg/ml in the absence of U118-MG cells. The bulk of these data suggested that Aβ₄₂ accumulation occurred in cocultures of H4 and U118-MG cells, although at much reduced levels compared to H4 cell cultures. It has been reported that glial cells can efficiently phagocyte Aβ by way of a series of endogenous receptors [64]. We investigated whether this property could explain the reduced levels of Aβ₄₂ production in coculture experiments. Results showed that Alexa Fluor 555-labeled Aβ₄₂ was rapidly taken up by U118-MG cells (Fig. 3B), suggesting that this mechanism was responsible for the low levels of detection of Aβ₄₂ in cocultures.

TNFα production by H4, U118-MG and cocultures in response to HSV-1 and Aβ treatments

Brain inflammation is a major component of AD [65 –67]. Levels of several cytokines (IL-1α, IL-1β, IL-6, TNFα, IFNα) have been reported to be increased in AD [67 –69]. We asked the question whether the in vitro model of coculture of neuronal and glial cells used in the present study generated cytokine production under various conditions, including exposure to HSV-1 and Aβ. In a first series of experiments, we assessed the production of pro-inflammatory cytokine TNFα in 16 h cultures. Results showed that H4 cells produced TNFα under basal conditions but production by U118-MG cells was significantly lower (Fig. 4A). TNFα production was similar to that of H4 cells in cocultures (Fig. 4A). Exposure of H4 cells to HSV-1 did not influence production of TNFα (Fig. 4B). Whereas the presence of Aβ₄₀ inhibited TNFαproduction in non-infected cells, it had no effect when H4 cells had been infected with HSV-1 (Fig. 4B). Similar observations were made in the case of Aβ₄₂, except that there was increased production of TNFα when cells were also exposed to HSV-1 (Fig. 4B). Interestingly, significantly increased production of TNFα was only observed when U118-MG cells (Fig. 4C) and cocultures (Fig. 4D) were grown in the combined presence of Aβ₄₂ and HSV-1.

IL-1β production by H4 and U118-MG cells and cocultures in response to HSV-1 and Aβ

H4 cells and cocultures of H4 and U118-MG cells produced the same amounts of pro-inflammatory IL-1β after 16 h, under basal conditions (Fig. 5A). However, U118-MG cells produced significantly less IL-1β under similar conditions (Fig. 5A). The presence of Aβ₄₀, Aβ₄₂, HSV-1, or a combination of thesechallenges did not affect production of IL-1β by H4 cells (Fig. 5B). In the case of U118-MG cells, results showed that HSV-1 in combination with or without Aβ₄₀ or Aβ₄₂ or, Aβ₄₂ alone potentiated production of IL-1β (Fig. 5C). However, only a combination of HSV-1 and Aβ₄₂ increased production of IL-1β in cocultures with respect to basal conditions (Fig. 5D).

INFα production in H4 and U118-MG cell lines and cocultures in response to HSV-1 and Aβ

Type 1 interferons contribute to neuroinflammation in AD and have been detected in brains of AD patients [70]. We investigated whether we could observe production of INFα in the in vitro model used in the present study. Results showed that H4 cells and cocultures of H4 and U118-MG cells produced low levels of INFα in 16 h cultures, under basal conditions, although there was absence of production in U118-MG cells (Fig. 6A). HSV-1 and a combination of HSV-1 and Aβ₄₀ or Aβ₄₂ significantly increased INFα production in H4 cells (Fig. 6B) but only Aβ₄₂, with or without HSV-1, showed this effect in U118-MG cells (Fig. 6C) and cocultures (Fig. 6D).

Inhibition of HSV-1 replication in de novo cultures of H4 cells by CM obtained from HSV-1-infected H4 cells

Previous work from our laboratory [55] and data presented in Fig. 2 showed that Aβ interfered with HSV-1 replication. CM from H4 cells infected with HSV-1 for 24 h was assayed in de novo cultures of H4 cells challenged with HSV-1. Results showed that the levels of HSV-1 replication were significantly lower (38.2 ± 11.1% , p < 0.01) (column #3) in comparison with de novo cultures exposed to HSV-1 without CM (column #1) or with medium from uninfected H4 cells (column #2) (Fig. 7A). These observations suggested that the inhibition of HSV-1 replication could be related to the presence of Aβ in CM (Fig. 7A). Further evidence that the presence of Aβ in CM was responsible for inhibition of HSV-1 replication was obtained by conducting similar experiments with fibroblast MRC-5 cells, a cell line that allows replication of HSV-1 [55] but that does not produce Aβ in response to HSV-1 (data not shown). As expected, HSV-1 replication occurred in these cells and levels of replication were not significantly different when the cells had been exposed to media from infected (CM) or uninfected MRC-5 cells (Fig. 7B).

Effects of BACE-1 inhibitor and type 1 IFN neutralizing antibodies on CM-dependent inhibition of HSV-1 replication in H4 cells

Data shown above suggested that Aβ present in CM was, at least in part, responsible for the inhibitory effect on HSV-1 replication in de novo cultures of H4 cells. This possibility was investigated by generating CM from H4 cells cultured in the absence or presence of a BACE-1 inhibitor. When infected H4 cells were treated with BACE-1 inhibitor, CM did not inhibit HSV-1 replication in de novo cultures of H4 cells (column 2, Fig. 8A), in marked contrastwith CM from infected cells not treated with BACE-1 (column 5, Fig 8A). Furthermore, exogenous addition of BACE-1 inhibitor to CM prior to de novo infection did not prevent inhibition by CM (columns #4 and #6, Fig. 8A). The bulk of these observations supported the interpretation that Aβ was the main component of CM responsible for inhibition of HSV-1 replication in the de novo cultures of H4 cells (Fig. 8A).

Type-1 IFN (INFα and IFNβ) have been reported to induce pro-inflammatory gene transcription and secretion of pro-inflammatory cytokines TNFα, IL-1β, and IL-6 [71]. Given the fact that these pro-inflammatory cytokines are produced by H4 cells, we asked whether type 1 IFN may be an additional factor in CM-associated inhibition of HSV-1 replication in de novo cultures of H4 cells exposed to HSV-1. Anti-IFNα or anti-IFNβ neutralizing antibodies were then added to CM prior to addition to de novo cultures of H4 cells 2 h prior to challenge (24 h) with HSV-1. Results showed that anti-IFNα or anti-IFNβ neutralizing antibodies did not prevent CM from inhibiting HSV-1 replication (Fig. 8B). BACE-1 inhibitor added to CM prior to assays did not prevent inhibition of HSV-1 replication (Fig. 8B), in further agreement with the suggested role of Aβ in these assays.

DISCUSSION

Overproduction of Aβ by neurons and/or their decreased degradation and clearance by microglia lead to their accumulation and neurotoxic effects associated with AD [36, 72]. Whether Aβ play a physiological role under normal healthy conditions remains to be established, although studies in animal models have suggested that Aβ may be involved in synaptic plasticity and memory [10, 73]. Furthermore, a protective role for Aβ as antimicrobial peptides in in vitro assays is slowly emerging [55 –57].

Neuroglioma H4 cells secreted Aβ₄₂ under basal conditions, in agreement with the fact that neurons are the major source of Aβ in the brain. Exposure of H4 cells to HSV-1 showed a steady increase in Aβ₄₂ production that was likely related to the viral load (Figs. 1A, B). These observations were in agreement with the recent reports that HSV-1 infection of neurons triggers AβPP processing in a rat model [74] and HSV-2 in human neuroblastoma cell cultures [75] and that HSV-1 infection causes cellular Aβ accumulation and secretase upregulation in vitro [76]. In contrast, H4 cells produced low levels of Aβ₄₀ even under challenge of HSV-1 (Figs. 1A, B). This result was unexpected given the fact that BACE-1 activity was detected under basal conditions and induced in cells infected with HSV-1 (Fig. 1C). U118-MG glioblastoma cells did not produce Aβ₄₀ and generated only low amounts of Aβ₄₂ even under conditions of HSV-1 infection (Fig. 1D). Further evidence that Aβ behaved as antimicrobial peptides and displayed anti-viral activity againstHSV-1 infection in vitro [55] was obtained by growing H4 cells over an extended period of time in the absence or presence of a BACE-1 inhibitor. Results conclusively showed a robust replication of HSV-1 in cells cultured in the presence of BACE-1 inhibitor but inhibition of viral replication in its absence (Fig. 2A), suggesting that Aβ production was responsible for inhibition of HSV-1 replication. This interpretation was supported by the findings of time-related inhibition of Aβ₄₂ production in H4 cultures maintained in the presence of BACE-1 inhibitor but not in its absence (Fig. 2B). We investigated whether a coculture of H4 and U118-MG cells would yield observations similar to those of the individual cell lines. Unexpectedly, results showed that production of Aβ₄₀ was barely detectable under basal conditions or when HSV-1 was present, suggesting that contact between the two cell lines did not upregulate production of Aβ₄₀ (Fig. 3A). Furthermore, levels of production of Aβ₄₂ were low under basal conditions and not upregulated in the presence of HSV-1 (Fig. 3A). These observations suggested that U118-MG cells had an inhibitory direct effect on Aβ₄₂-producing H4 cells or that U118-MG cells captured de novo-produced Aβ₄₂ due to their phagocytic properties, or both. Interpretation for the first possibility was considered unlikely on the basis of physiological considerations. For instance, microglial cells play a protective role in capture and degradation of Aβ under healthy homeostatic conditions, thus preventing undesired accumulation of Aβ and their neurotoxic effects [36, 64]. It was therefore considered unlikely that the presence of U118-MG cells would prevent Aβ production by H4 cells. However, incubation of U118-MG cells with AlexaFluor 555-labeled Aβ₄₂ clearly showed that it was rapidly internalized, suggesting that this mechanism was responsible for the low levels of Aβ production in cocultures (Fig. 3B).

Neuroinflammation triggered by infection or other insults to the brain activate glial cells to secrete the pro-inflammatory cytokines TNFα, IL-1β, and IL-6 [35, 77]. We investigated how the coculture model used here would behave in response to HSV-1 infection and the presence of exogenous Aβ with respect to pro-inflammatory cytokine production. Results clearly indicated that Aβ₄₂ alone or in combination withHSV-1 upregulated production of IL-1β, TNFα, and IFNα in the coculture model. These observations were in agreement with an expected behavior of glial cells in response to infection and Aβ₄₂ recognition which triggers a pro-inflammatory response involved in AD. Upregulation of type 1 interferons has been reported in postmortem AD brains and in wild type cultures of neurons challenged with Aβ₄₂ [70]. Here, INFα was produced by H4 cells and cocultures under basal conditions (Fig. 6A). Aβ did not upregulate INFα production in H4 cells (Fig. 6B). This observation was in disagreement with that reported [70] and may be related to the use of a neuroglioma cell line as opposed to wild type neurons. Importantly, a combination of HSV-1 and Aβ stimulated INFα production (Fig. 6B) has been reported in various other neuronal cell lines [78, 79]. These data supported the interpretation that the modulation of this pro-inflammatory (TNFα, IL-1) and anti-viral (IFNα) response may contribute to the development of HSV-1 infection latency in humans at an immunoprivileged site but without any neuronal destruction. This inflammatory response may contribute to maintain latent HSV-1 infection [80].

We asked the question whether the anti-viral protective effect of Aβ [55] could be demonstrated using HSV-1-sensitive, Aβ-producing H4 cells. For obvious reasons, the coculture model could not be used because U118-MG cells rapidly internalized Aβ (Fig. 3B). CM from cultures of Aβ-producing H4 cells was harvested after 24 h, at a time point where HSV-1 replication was low (Fig. 2A). Results showed that CM inhibitedHSV-1 replication in de novo cultures of H4 cells (Fig. 7). CM from HSV-1-sensitive fibroblast MRC-5 cells was prepared under similar conditions and assayed for inhibition of HSV-1 replication in de novo cultures. As expected, these CM did not show anti-viral activity in contrast to Aβ-producing H4 cells (Fig. 7A, column #3 and Fig. 8A, columns #5 and #6). The bulk of our findings strongly suggested that inhibition of HSV-1 replication in de novo cultures of HSV-1-infected H4 cells was due to the presence of Aβ produced by H4 cells (Fig. 1). Data in support of this interpretation were obtained by preparing CM from H4 cells under various conditions and assaying them in de novo cultures of H4 cells. Data clearly showed that inhibition of Aβ production due to the presence of BACE-1 inhibitor in the cultures used to prepare CM prevented this CM from inhibiting HSV-1 replication (Fig. 8A, column 2). In marked contrast, CM from BACE-1-free cultures significantly inhibited HSV-1 replication (Fig. 8A, column 5). However, the data did not exclude the possibility that type 1 IFN was involved in inhibition of HSV-1 replication. This possibility was investigated by adding anti-IFNα and/or anti-IFNβ neutralizing antibodies to CM and then infecting de novo cultures with HSV-1. Results showed that the presence of these neutralizing antibodies did not prevent CM from inhibiting HSV-1 replication in de novo cultures (Fig. 8B), suggesting that extracellular type 1 IFN was not involved in inhibition of HSV-1 replication. However, our data did not formally exclude the possibility that intracellularly produced type I IFN could contribute to reduction of infectious virus production.

Although based on a minimal cellular model of cocultures of neuronal and glial cells, the bulk of our results supported our previous observations [55] and those of White et al. [56] with respect to Aβ possessing anti-viral activity in vitro and, presumably, in the CNS. Production of Aβ may represent an initial attempt to set in motion a defensive brain response to curtail viral and, possibly, other aggressions. In this respect, it has been shown that infection by HSV-1 triggers the release of Aβ by human and rat neurons [81, 82]. Therefore our work further contributes to our understanding of the effect of HSV-1 on neuronal cells, and the induction of Aβ production. Furthermore, data presented here showed that Aβ production, even in cell cultures, is at sufficient levels to exert a beneficial anti-viral effect. Neurons bathe in much smaller volumes of extracellular medium in the brain and it is therefore likely that Aβ production provides effective protection. To the best of our knowledge, data presented here are the first to establish a link between neuronal Aβ production and its anti-viral role as a result of HSV-1 infection and, its putative role in AD pathogenesis. Furthermore, our results showed a sensitive regulatory interaction by microglia on Aβ production by neuronal cells, playing either protective or detrimental role for neuroinflammation/degeneration probably at different stages of the disease. Our observations may extend to protection against other inflammatory agents but this possibility needs further investigation. However, the negative facet of Aβ-dependent defense mechanism is that sustained induction of Aβ production in the brain in the case of frequent reactivation of HSV-1, may overwhelm Aβ clearance and that would be detrimental to brain function. This situation becomes particularly critical with aging as microglia become less efficient at eliminating viruses and Aβ [83]. Aβ overproduction and deposit in amyloid plaques triggers a state of inflammation that is associated with neuronal loss and region-specific destruction of the aging brain and progressive development of AD.

Taking into account the putative novel role of Aβ as antimicrobial peptides in the CNS, a model can be suggested to summarize AD pathophysiology in relationship to viral infection by members of the Herpesviridae family and influenza and, other microbial infections of the brain. According to a working model (Fig. 9), Aβ are normally produced by neurons under homeostatic conditions at a rate required to fulfill their physiological functions in maintaining synaptic plasticity and memory [73], as well as antimicrobial protection. A balance between Aβ production and regulation against overproduction by microglia-dependent phagocytosis would maintain a homeostatic condition (Fig. 9, physiological state). However, in cases of brain trauma such as infections, hypoxia at birth, metabolic defects, misfolded proteins and other insults, a protective mechanism would be triggered that would involve increased Aβ production. Neuron infection by members of the Herpesviridae family is a condition that affects over 90% of the world’s population at least in its latent, asymptomatic, form. HSV-1 reactivation under conditions of weakened immune response or stress [84] would trigger increased Aβ production that would then induce microglia activation and release of pro-inflammatory cytokines (Fig. 9, pathological state) on an individual basis. Pro-inflammatory cytokines would act as paracrine mediators, sustaining microglia and astrocytic activation [83] and initiating a vicious circle of inflammatory responses. In addition, pro-inflammatory cytokines could cross the blood brain barrier, initiating systemic inflammation [31, 66]. Overproduction and accumulation of Aβ would interfere with their homeostatic physiological functions, would induce damage to neurons to which Aβ oligomers will associate in a fibrillar form and would generate senile plaques. Recurrence of this series of events during a lifetime would obviously result in amplification and irreversible damage to specific regions of the brain, namely the hippocampus and temporal lobes, which have been reported to be a region of HSV-1 location [44] and a major site of injury in AD.

In conclusion, our data have clearly shown that Aβ production by a model of neurons was triggered by HSV infection. It is conceivable that Aβ production could be harmful under situations of frequent reactivation of HSV-1 in the brain throughout life. Repeated reinfections and subsequent elevated production of Aβ are involved in microglia and astrocytic activation, which are deleterious for the brain. Our data reinforce the recent thinking that Aβ may be beneficial and not simply a byproduct in the pathogenesis of AD. It may be suggested that therapeutic agents should target the aggressors that induce Aβ production such as HSV-1 infection rather than Aβ because of its frequent detection in the brains of AD patients.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from Canadian Institutes of Health Research (CIHR) (No. 106634), the Université de Sherbrooke, the Société des Médecins de l’Université de Sherbrooke (SMUS) and the Research Center on Aging.

Authors’ disclosures available online ().

References

Ballard

, Gauthier

, Corbett

, Brayne

, Aarsland

, Jones

(2011) Alzheimer’s disease. Lancet 377, 1019–1031.

Castellani

, Rolston

, Smith

(2010) Alzheimerdisease. Dis Mon 56, 484–546.

Tam

JHK

, Pasternak

(2012) Amyloid and Alzheimer’s disease: Inside and out. Can J Neurol Sci 9, 286–298.

World Health Organization report.(2013) Dementia: A public health priority. 11 April 2013.

Kern

, Behl

(2009) The unsolved relationship of brain aging and late-onset Alzheimer disease. Biochim Biophys Acta 10, 1124–1132.

Karran

, Mercken

, De Strooper

(2011) The amyloid cascade hypothesis for Alzheimer’s disease: An appraisal for the development of therapeutics. Nature Rev Drug Discov 10, 698–712.

Seeman

, Seeman

(2011) Alzheimer’s disease: β-amyloid plaque formation in human brain. Synapse 65, 1289–1297.

Claeysen

, Cochet

, Donneger

, Dumuis

, Bockaert

, Giannoni

(2012) Alzheimer culprits: Cellular crossroads and interplay. Cell Signal 24, 1831–1840.

LaFerla

, Green

, Oddo

(2007) Intracellular amyloid-β in Alzheimer’s disease. Nature Rev Neurosci 8, 499–509.

10.

Pearson

, Peers

(2006) Physiological roles for amyloid beta peptides. J Physiol 575, 5–10.

11.

Rivest

(2009) Regulation of innate immune responses in the brain. Nature Rev Immunol 9, 429–439.

12.

Griciuc

, Serrano-Pozo

, Parrado

, Lesinski

, Asselin

, Mullin

, Hooli

, Choi

, Hyman

, Tanzi

(2013) Alzheimer’s disease risk gene CD33 inhibits microglial uptake of amyloid beta. Neuron 78, 631–643.

13.

Bagyinszky

, Youn

, An

, Kim

(2014) The genetics of Alzheimer’s disease. Clin Interv Aging 9, 535–551.

14.

Wang

, He

, Wang

(2015) Association between polymorphisms of the insulin-degrading enzyme gene and late-onset Alzheimer disease. J Geriatr Psychiatry Neurol 28, 94–108.

15.

De la Monte

, Tong

(2014) Brain metabolic dysfunction at the core of Alzheimer’s disease. Biochem Pharmacol 88, 548–559.

16.

Morgen

, Frölich

(2015) The metabolism hypothesis of Alzheimer’s disease: From the concept of central insulin resistance and associated consequences to insulin therapy. J Neural Transm 122, 499–504.

17.

Baglio

, Saresella

, Preti

, Cabinio

, Griffanti

, Marventano

, Piancone

, Calabrese

, Nemni

, Clerici

(2013) Neuroinflammation and brain functional disconnection in Alzheimer’s disease. Front Aging Neurosci 5, 81.

18.

Morales

, Guzmán-Martínez

, Cerda-Troncoso

, Farías

, Maccioni

(2014) Neuroinflammation in the pathogenesis of Alzheimer’s disease. A rational framework for the search of novel therapeutic approaches. Front Cell Neurosci 8, 112.

19.

Zhang

, Jiang

(2015) Neuroinflammation in Alzheimer’s disease. Neuropsychiatr Dis Treat 11, 243–256.

20.

Armstrong

(2013) What causes Alzheimer’s disease? Folia Neuropathol 51, 169–188.

21.

De Chiara

, Marcocci

, Sgarbanti

, Civitelli

, Ripoli

, Piacentini

, Garaci

, Grassi

, Palamara

(2012) Infectious Agents and Neurodegeneration. Mol Neurobiol 46, 614–638.

22.

Miklossy

(2011) Emerging roles of pathogens in Alzheimer disease. Expert Rev Mol Med 13, e30.

23.

Fulop

, Lacombe

, Cunnane

, Le Page

, Dupuis

, Frost

, Bourgade-Navarro

, Goldeck

, Larbi

, Pawelec

(2013) Elusive Alzheimer’s disease: Can immune signatures help our understanding of this challenging disease? Part 1: Clinical and historical background. Discov Med 15, 23–32.

24.

Reitz

, Mayeux

(2014) Alzheimer disease: Epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem Pharmacol 88, 640–651.

25.

Bekris

, Galloway

, Millard

, Lockhart

, Li

, Galasko

, Farlow

, Clark

, Quinn

, Kaye

, Schellenberg

, Leverenz

, Seubert

, Tsuang

, Peskind

, Yu

(2010) Amyloid precursor protein (APP) processing genes and cerebrospinal fluid APP cleavage product levels in Alzheimer’s disease. Neurobiol Aging 32, 13–23.

26.

Franceschi

, Bonafè

, Valensin

, Olivieri

, De Luca

, Ottaviani

, De Benedictis

(2000) Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 908, 244–254.

27.

Larbi

, Pawelec

, Witkowski

, Schipper

, Derhovanessian

, Goldeck

, Fulop

(2009) Dramatic shifts in circulating CD4 but not CD8 T cell subsets in mild Alzheimer’s disease. J Alzheimers Dis 17, 91–103.

28.

Larbi

, Fortin

, Dupuis

, Berrougui

, Khalil

, Fulop

(2014) Immunomodulatory role of high-density lipoproteins: Impact on immunosenescence. Age 36, 9712.

29.

Streit

, Xue

(2014) Human CNS immune senescence and neurodegeneration. Curr Opin Immunol 29, 93–96.

30.

Cai

, Hussain

, Yan

(2014) Microglia, neuroinflammation, and beta-amyloid protein in Alzheimer’s disease. Int J Neurosci 124, 307–321.

31.

Le Page

, Bourgade

, Lamoureux

, Frost

, Pawelec

, Larbi

, Witkowski

, Dupuis

, Fülöp

(2015) NK cells are activated in amnestic mild cognitive impairment but not in mild Alzheimer’s disease patients. J Alzheimers Dis 46, 93–107.

32.

Takeda

, Sato

, Morishita

(2014) Systemic inflammation, blood-brain barrier vulnerability and cognitive/non-cognitive symptoms in Alzheimer disease: Relevance to pathogenesis and therapy. Front Aging Neurosci 6, 171.

33.

Zhang

, Miller

, Madison

, Jin

, Honrada

, Harris

, Katz

, Forshew

, McGrath

(2013) Systemic immune system alterations in early stages of Alzheimer’s disease. J Neuroimmunol 256, 38–42.

34.

Angelova

, Abramov

(2014) Interaction of neurons and astrocytes underlies the mechanism of Aβ-induced neurotoxicity. Biochem Soc Trans 42, 1286–1290.

35.

Glass

, Saijo

, Winner

, Marchetto

, Gage

(2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934.

36.

Doens

, Fernández

(2014) Microglia receptors and their implications in the response to amyloid β for Alzheimer’s disease pathogenesis. J Neuroinflammation 11, 48.

37.

Benilova

, Karran

, De Strooper

(2012) The toxic Aβ oligomer and Alzheimer’s disease: An emperor in need of clothes. Nat Neurosci 15, 349–357.

38.

Armstrong

(2014) A critical analysis of the ‘amyloid cascade hypothesis’. Folia Neuropathol 52, 211–225.

39.

Castello

, Soriano

(2014) On the origin of Alzheimer’s disease. Trials and tribulations of the amyloid hypothesis. Ageing Res Rev 13, 10–12.

40.

Morris

, Clark

, Vissel

(2014) Inconsistencies and controversies surrounding the amyloid hypothesis of Alzheimer’s disease. Acta Neuropathol Commun 2, 135.

41.

Reitz

(2012) Alzheimer’s disease and the amyloid cascade hypothesis: A critical review. Int J Alzheimers Dis 2012, 369808.

42.

Sorrentino

, Iuliano

, Polverino

, Jacini

, Sorrentino

(2014) The dark sides of amyloid in Alzheimer’s disease pathogenesis. FEBS Lett 588, 641–652.

43.

Piacentini

, De Chiara

, Li Puma

, Ripoli

, Marcocci

, Garaci

, Palamara

, Grassi

(2014) HSV-1 and Alzheimer’s disease: More than a hypothesis. FrontPharmacol 5, 97.

44.

Wozniak

, Mee

, Itzhaki

(2009) Herpes simplex virus type 1 DNA is located within Alzheimer’s disease amyloid plaques. J Pathol 217, 131–138.

45.

Letenneur

, Pérès

, Fleury

, Garrigue

, Barberger-Gateau

, Helmer

, Orgogozo

, Gauthier

, Dartigues

(2008) Seropositivity to herpes simplex virus antibodies and risk of Alzheimer’s disease: A population-based cohort study.e. PLoS One 3, 3637.

46.

Lin

, Wozniak

, Cooper

, Wilcock

, Itzhaki

(2002) Herpesviruses in brain and Alzheimer’s disease. J Pathol 197, 395–402.

47.

Steiner

, Benninger

(2013) Update on herpes virus infections of the nervous system. Curr Neurol Neurosci Rep 13, 414.

48.

Beffert

, Bertrand

, Champagne

, Gauthier

, Poirier

(1998) HSV-1 in brain and risk of Alzheimer disease. Lancet 351, 1330–1331.

49.

Itzhaki

, Lin

, Dhang

, Wilcock

, Fragher

, Jamieson

(1997) Herpes simplex virus type 1 in brain and risk of Alzheimer’s disease. Lancet 349, 241–247.

50.

Held

, Derfuss

(2011) Control of HSV-1 latency in human trigeminal ganglia–current overview. J Neurovirol 17, 518–527.

51.

Itzhaki

, Wozniak

(2006) Herpes simplex virus type 1, apolipoprotein E, and cholesterol: A dangerous liaison in Alzheimer’s disease and other disorders. Prog Lipid Res 45, 73–90.

52.

Denaro

, Staub

, Colmer

, Freed

(2003) Coexistence of Alzheimer disease neuropathology with herpes simplex encephalitis. Cell Mol Biol 49, 1233–1240.

53.

Féart

, Helmer

, Fleury

, Béjot

, Ritchie

, Amouyel

, Schraen-Maschke

, Buée

, Lambert

, Letenneur

, Dartigues

(2011) Association between IgM anti-herpes simplex virus and plasma amyloid-beta levels. PLoS One 6, e29480.

54.

Mancuso

, Baglio

, Agostini

, Cabinio

, Laganá

, Hernis

, Margaritella

, Guerini

, Zanzottera

, Nemni

, Clerici

(2014) Relationship between herpes simplex virus-1-specific antibody titers and cortical brain damage in Alzheimer’s disease and amnestic mild cognitive impairment. Front Aging Neurosci 6, 285.

55.

Bourgade

, Garneau

, Giroux

, Le Page

, Bocti

, Dupuis

, Frost

, Fülöp

Jr (2015) β-Amyloid peptides display protective activity against the human Alzheimer’s disease-associated herpes simplex virus-1. Biogerontology 16, 85–98.

56.

White

, Kandel

, Tripathi

, Condon

, Qi

, Taubenberger

, Hartshorn

(2014) Alzheimer’s associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes. PLoS One 9, e101364.

57.

Soscia

, Kirby

, Washicosky

, Tucker

, Ingelsson

, Hyman

, Burton

, Goldstein

, Duong

, Tanzi

, Moir

(2010) The Alzheimer’s disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS One 5, e9505.

58.

Arispe

, Diaz

, Simakova

(2007) Aβ ion channel. Prospects for treating Alzheimer’s disease with Aβ channel blockers. Biochim Biophys Acta 1768, 1952–1965.

59.

Deshpande

, Mina

, Glabe

, Busciglio

(2006) Diffetent conformations of Amyloid β induce neurotoxicity by distinct mechanisms in human cortical neurons. J Neurosci 26, 6011–6018.

60.

Reyes Barcelo

, Gonzalez-Velasquez

, Moss

(2009) Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells. J Biol Eng 3, 5.

61.

Lue

, kuo

, Roher

, Brachova

, Shen

, Sue

, Beach

, Kurth

, Rydel

, Rogers

(1999) Soluble amyloid β peptide concentration as a predictor of synaptic change in Alzheimer’s disease. Am J Pathol 3, 853–862.

62.

Vingataramin

, Frost

(2015) A single protocol for extraction of gDNA from bacteria and yeast. Biotechniques 58, 120–125.

63.

Williams

, Serpell

(2011) Membrane and surface interactions of Alzheimer’s Aβ peptide–insights into the mechanism of cytotoxicity. FEBS J 278, 3905–3917.

64.

Lee

, Landreth

(2010) The role of microglia in amyloid clearance from the AD brain. J Neural Transm 117, 949–960.

65.

Heneka

, Carson

, El Khoury

, Landreth

, Brosseron

, Feinstein

, Jacobs

, Wyss-Coray

, Vitorica

, Ransohoff

, Herrup

, Frautschy

, Finsen

, Brown

, Verkhratsky

, Yamanaka

, Koistinaho

, Latz

, Halle

, Petzold

, Town

, Morgan

, Shinohara

, Perry

, Holmes

, Bazan

, Brooks

, Hunot

, Joseph

, Deigendesch

, Garaschuk

, Boddeke

, Dinarello

, Breitner

, Cole

, Golenbock

, Kummer

(2015) Neuroinflammation in Alzheimer’s disease. Lancet Neurol 4, 388–405.

66.

Rubio-Perez

, Morillas-Ruiz

(2012) A review: Inflammatory process in Alzheimer’s disease, role of cytokines. ScientificWorldJournal 2012, 756357.

67.

Zhang

, Jiang

(2015) Neuroinflammation in Alzheimer’s disease. Neuropsychiatr Dis Treat 11, 243–256.

68.

Lee

, Chung

, Choi

, Suh

, Oh

, Hong

(2009) Peripheral cytokines and chemokines in Alzheimer’s disease. Dement Geriatr Cogn Disord 28, 281–287.

69.

McGeer

, McGeer

(1997) Inflammatory cytokines in the CNS. CNS Drugs 7, 214–287.

70.

Taylor

, Minter

, Newman

, Zhang

, Adlard

, Crack

(2014) Type-1 interferon signaling mediates neuro-inflammatory events in models of Alzheimer’s disease. Neurobiol Aging 35, 1012–1023.

71.

de Weerd

, Nguyen

(2012) The interferons and their receptors - distribution and regulation. Immunol Cell Biol 90, 483–491.

72.

, Ye

(2015) Microglial Aβ receptors in Alzheimer’s disease. Cell Mol Neurobiol 35, 71–83.

73.

Puzzo

, Arancio

(2013) Amyloid-β peptide: Dr. Jekyll or Mr. Hyde? J Alzheimers Dis 33, S111–S120.

74.

Civitelli

, Marcocci

, Celestino

, Piacentini

, Garaci

, Grassi

, De Chiara

, Palamara

(2015) Herpes simplex virus type 1 infection in neurons leads to production and nuclear localization of APP intracellular domain (AICD): Implications for Alzheimer’s disease pathogenesis. J Neurovirol 21, 480–490.

75.

Kristen

, Santana

, Sastre

, Recuero

, Bullido

, Aldudo

(2015) Herpes simplex virus type 2 infection induces AD-like neurodegeneration markers in human neuroblastoma cells. Neurobiol Aging 36, 2737–2747.

76.

Wozniak

, Itzhaki

, Shipley

, Dobson

(2007) Herpes simplex virus infection causes cellular beta-amyloid accumulation and secretase upregulation. Neurosci Lett 429, 95–100.

77.

Lee

, Han

, Nam

, Oh

, Hong

(2010) Inflammation and Alzheimer’s disease. Arch Pharm Res 33, 1539–1556.

78.

Gianni

, Leoni

, Campadelli-Fiume

(2013) Type I interferon and NF-κB activation elicited by herpes simplex virus gH/gL via αvβ3 integrin in epithelial and neuronal cell lines. J Virol 87, 13911–13916.

79.

Préhaud

, Mégret

, Lafage

, Lafon

(2005) Virus infection switches TLR-3-positive human neurons to become strong producers of beta interferon. J Virol 79, 12893–12904.

80.

Theil

, Derfuss

, Paripovic

, Herberger

, Meinl

, Schueler

, Strupp

, Arbusow

, Brandt

(2003) Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response. Am J Pathol 163, 2179–2184.

81.

De Chiara

, Marcocci

, Civitelli

, Argnani

, Piacentini

, Ripoli

, Manservigi

, Grassi

, Garaci

, Palamara

(2010) APP processing induced by herpes simplex virus type 1 (HSV-1) yields several APP fragments in human and rat neuronal cells. PLoS One 5, e13989.

82.

Piacentini

, Civitelli

, Ripoli

, Marcocci

, De Chiara

, Garaci

, Azzena

, Palamara

, Grassi

(2011) HSV-1 promotes Ca2+-mediated APP phosphorylation and Aβ accumulation in rat cortical neurons. Neurobiol Aging 32, e13–e26.

83.

Solito

, Sastre

(2012) Microglia function in Alzheimer’s disease. Front Pharmacol 3, 14.

84.

Nicoll

, Proença

, Efstathiou

(2012) The molecular basis of herpes simplex virus latency. FEMS Microbiol Rev 36, 684–705.

Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Cell cultures and cocultures

Synthetic amyloid-β peptides, antibodies, and BACE-1 inhibitor

HSV-1

Quantification of Aβ by ELISA

Measure of BACE-1 activity

Cytokine quantification

DNA isolation and amplification of cells treated with Aβ and HSV-1

Analysis of Alexa Fluor 555-labeled Aβ42 uptake by U118-MG cells using fluorescence microscopy

Cell viability

Statistical analysis

RESULTS

Time- and β-secretase-dependent production of Aβ in H4 neuroglioma cells and U118-MG glioblastoma cells in response to HSV-1 infection

Effect of β-secretase inhibition on HSV-1 replication and Aβ42 production in long-term cultures of H4 cells

Aβ production in cocultures of H4 and U118-MG cells in response to HSV-1 infection

TNFα production by H4, U118-MG and cocultures in response to HSV-1 and Aβ treatments

IL-1β production by H4 and U118-MG cells and cocultures in response to HSV-1 and Aβ

INFα production in H4 and U118-MG cell lines and cocultures in response to HSV-1 and Aβ

Inhibition of HSV-1 replication in de novo cultures of H4 cells by CM obtained from HSV-1-infected H4 cells

Effects of BACE-1 inhibitor and type 1 IFN neutralizing antibodies on CM-dependent inhibition of HSV-1 replication in H4 cells

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Analysis of Alexa Fluor 555-labeled Aβ₄₂ uptake by U118-MG cells using fluorescence microscopy

Effect of β-secretase inhibition on HSV-1 replication and Aβ₄₂ production in long-term cultures of H4 cells