Anti-Viral Properties of Amyloid-β Peptides

Abstract

Amyloid-β (Aβ) peptides generated by the amyloidogenic pathway of amyloid-β protein precursor processing contribute significantly to neurodegeneration characteristic of Alzheimer’s disease (AD). The involvement of Aβ peptides in the etiology of AD remains a subject of debate. Data published in the last 6 years by three different groups have added a new twist by revealing that Aβ peptides could act as antimicrobial peptides (AMP) in in vitro assays against some common and clinically relevant microorganisms, inhibit replication of seasonal and pandemic strains of influenza A and HSV-1 virus. These observations are of significance with respect to the notion that pathogens may be important contributors to the development of AD, particularly in the case of herpes simplex virus (HSV) infection, which often resides in the same cerebral sites where AD arises. Here, we review the data that support the interpretation that Aβ peptides behave as AMP, with an emphasis on studies concerning HSV-1 and a putative molecular mechanism that suggests that interactions between Aβ peptides and the HSV-1 fusogenic protein gB lead to impairment of HSV-1 infectivity by preventing the virus from fusing with the plasma membrane. A number of avenues for future research are suggested.

Keywords

Alzheimer’s disease amyloid-beta peptides antimicrobial peptides antiviral activity cocultures glycoprotein B herpes simplex virus influenza virus membrane proximal region

INTRODUCTION

In 1907, Alois Alzheimer described a female patient who presented unusual symptoms of dementia [1]. Postmortem examination of the brain of this patient with extensive cognitive deterioration revealed the presence of cortical atrophy associated with senile plaques and neurofibrillary tangles. This single historical case description has had far-reaching consequences in what has become known as Alzheimer’s disease (AD). AD is now recognized as the most common form of dementia in the world [2, 3]. It is a progressive neurodegenerative disorder that is characterized by irreversible neuronal degeneration in specific regions of the brain, especially the neocortex and the hippocampus, which is the seat of memory [4, 5]. The clinical manifestations of AD are an initial decline in short term memory that progresses over the years to complete loss accompanied by impaired language skills, alterations of cognitive functions including rational judgment and decision making, a loss of self-autonomy and, in a large number of cases, uncontrolled and aggressive behavioral disturbances [6 –8]. AD has become a growing public health concern. Surveys estimate that AD currently affects over 47 million patients worldwide and projections for the next decades are staggering. According to conservative models of AD epidemiology, progression from early diagnosis to full-blown disease is projected to affect over 65 million individuals in 2030 and more than 131.5 millions by the year 2050 [9, 10]. Collateral costs put a severe burden not only on the affected individuals but also on families, professional caretakers, specialized home care institutions and society in general. From a standpoint of monetary costs, global estimates of the financial expenditure in 2010 exceeded 600 billion US dollars [11] and will likely reach trillions of dollars if current projections are accurate. This paramount medical and social problem is further compounded by the general increase in life expectancy and the fact that aging is the primary risk factor, at least in the age-related form of AD [12 –16]. Despite extensive efforts documented by more than 116,854 references to AD currently archived in PubMed^® and 7,076 references to its prevention at the moment of writing, there is still no efficient treatment to halt progression of the disease, let alone a cure for it [17 –19]. AD is a highly complex and debilitating disease that manifests as an overall deterioration of the human condition and brings irrevocable earlier death, in nearly all cases [20]. Clinical interventions have targeted general and neuropsychiatric symptoms by using cholinergic inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists and behavior control drugs, inhibition of production of fragments of amyloid-β protein precursor (AβPP) processing [21], anti-inflammatory drugs [22, 23], as well as medications that target metabolic aberration products associated with AD [6 , 25]. These multi-targeted approaches are reflected by the fact that the underlying causes of AD have not been clearly established, making efficient treatment highly difficult [26]. Aside from the obvious relationship to aging, particularly in the case of the late onset form of the disease, several possibilities have been put forward as risk-associated conditions. These have been recently reviewed [27]. They include environmental factors [28], head injury [26, 29], malnutrition [30, 31], structural changes in the vasculature [32 –34], alterations of the cholinergic and cortico-cortical pathways [35, 36], genetic factors [37 –39], alterations in immune functions [40 –42], mitochondrial dysfunction [2, 43], altered blood-brain barrier [44], pathogen and virus infections [45 –50], and local and systemic inflammation [51 –54].

EARLY AND LATE FORMS OF AD

There are two clinical forms of AD, according to their characteristic pathogenesis and the time of onset. One form of the disease is referred to as the early onset AD (EOAD) and it corresponds to the genetic and familial form of the disease. The late onset 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 and deposits of cortical senile plaques and formation of neurofibrillary tangles in the brain of affected patients [20, 55]. EOAD represents 5% to 10% of documented AD cases whereas LOAD accounts for the remaining number of cases. Three genes involved in amyloid-β protein precursor (AβPP) metabolism are considered the main risk factors for EOAD: AβPP itself and AβPP-processing proteases presenilin 1 (PSEN1) and presenilin 2 (PSEN2) [56]. AβPP is a single-pass type 1 transplasma membrane protein [57] that is expressed in the central nervous system (CNS) as well as most somatic tissues [58, 59]. Although the physiological role of AβPP is still not clear, the bulk of data collected so far suggests that AβPP may be involved as a trophic factor to provide help for neurite outgrowth and synaptogenesis [60, 61], especially in the developing brain and to play a role in neuronal signaling functions [62, 63]. Expression of AβPP is influenced by trauma to the brain. For instance, its expression is upregulated in AD [64] and following brain injury, in which case it may be essential to participate to restore synaptic function [65]. At least 25 pathological mutations in AβPP have been associated with EOAD [2, 66].

So far, no gene has been identified as the cause of LOAD. However, environmental factors, family history, diabetes mellitus, educational status, hypertension, hypercholesterolemia, brain infection, and head trauma have been suggested as risk factors that may contribute to LOAD [15 , 67–69]. Furthermore, mutations/variants of a number of genes have shown strong association as risk factors of LOAD. Among these, inheritance of the apolipoprotein E ɛ4 (APOE ɛ4) allele appears to be the most prominent candidate [56 , 70–73]. However, genome-wide association studies have also identified medium-to-low risk gene products such as triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor expressed on a variety of cells including microglia [74, 75], phospholipase D3 (PLD3), a widely expressed phospholipase for triglyceride metabolism, as well as a large number of gene products associated with immune response, cell physiology and epigenetics [25], and several other candidate genes [56, 76].

PROCESSING OF THE AMYLOID-β PROTEIN PRECURSOR

Proteolytic fragments generated from extracellular and intracellular portions of the molecule

The gene coding for AβPP is located on chromosome 21 and is interspersed with 18 exons. Alternative splicing of gene transcripts results in several isoforms of AβPP. The AβPP695 isoform lacks the gene sequence from exons 7 and 8. This is the isoform preferentially expressed in neurons [77, 78]. AβPP is proteolytically processed by two competing pathways (Fig. 1). One pathway gives rise to amyloid-β (Aβ) peptides and other fragments and is called the amyloidogenic pathway. The other pathway does not generate Aβ peptides but fragments of a different structure and is called the non-amyloidogenic pathway. In the non-amyloidogenic pathway, AβPP is cleaved by α-secretases (ADAM proteases/TACE) to generate a large extracellular soluble secreted fragment (sAβPPα) and the plasma membrane-associated αAβPP-CTF fragment of 83 amino acid residues (C83). C83 is further cleaved by γ-secretase [79] to release a P3 fragment and the AβPP intracellular domain (AICD). Whereas the physiological role of αAβPP-CTF has not been clearly established, AICD may translocate to the nucleus and play a role in the transcription of AβPP [80, 81]. In the case of the amyloidogenic pathway, AβPP is internalized into endocytic compartments (Fig. 1B) where it is cleaved into two fragments termed sAβPPβ and AβPP-C99, as the result of the proteolytic activity of the β-secretase BACE. AβPP-99 is then cleaved by a γ-secretase complex comprising presenilin 1 to generate AICD and Aβ peptides, which are secreted [82]. The major isoforms of Aβ peptides are composed respectively of 38 (Aβ₃₈, <20%), 40 (Aβ₄₀, <80%), and 42 (Aβ₄₂, ≈ 10%) amino acid residues [83]. Although similar in molecular size, Aβ₄₀ and Aβ₄₂ differ in their physical properties. For instance, hydrophobicity and propensity to oligomerize into a fibrillar form and cytotoxicity are mostly associated with the Aβ₄₂ peptide [84 –87].

Aβ peptides and microtubule-associated tau protein solubility is the key to a healthy brain

Extracellular formation of proteolysis-resistant insoluble fibrils of Aβ peptides that deposit in senile plaques and, intracellular neurofibrillary tangles resulting from hyperphosphorylation of the microtubule-associated protein tau, are the neuropathological hallmarks of AD [6 , 86–90]. Tau is an essential component of microtubules, which are one of the fundamental elements of the cytoskeleton involved in anterograde and retrograde transport of vesicles, space distribution of mitochondria, and chromosome partitioning during cell division. In AD, tau can form insoluble fibrils that deposit inside the cell [91]. Human tau is encoded by the microtubule-associated protein tau gene, MAPT, that comprises 16 exons and which gives rise to 6 isoforms [92]. The longest form of tau comprises 441 amino acid residues and the shortest, 352. In the brain, tau is mainly found in neurons but is also present at low levels in glia1 cells. Tau can undergo a large number of post-transcriptional modifications that include phosphorylation, glycosylation, deamidation, and acetylation, among others [93]. Tau is a highly hydrophilic protein since the longest form contains 80 hydrophilic amino acid residues and 114 polar (acidic and basic) amino acid residues. Therefore, tau is a soluble protein and it is expected that phosphorylation would favor its water solubility. In this respect, physiological tau is phosphorylated and this is an essential modification required for its functions, namely its associative role with microtubules and microtubule-associated proteins [94]. However, it is abnormally hyperphosphorylated in the AD brain [95 –99]. This observation has been taken as evidence for its neurotoxicity [91] and shown to be a reliable feature of AD. It has been suggested that hyperphosphorylated tau may be the major culprit in the pathology of AD [91]. It is still unclear how hyperphosphorylated tau assembles into intracellular (and extracellular) insoluble fibrillary structures and how this behavior relates to the mechanism of its neurotoxicity.

Aβ are normal products of AβPP processing, although the mechanism that favors the non-amyloidogenic over the amyloidogenic pathway remains under investigation. Aβ₄₀ and Aβ₄₂ peptides differ by only two amino acid residues at the C-terminal but they display intrinsic physical differences. For instance, Aβ₄₂ is more hydrophobic and more cytotoxic than its Aβ₄₀ counterpart [85 , 101]. These structure-related features that may be due to its properties to be more prone to aggregate than Aβ₄₀, are due to the presence of the additional two aliphatic (Ile and Ala) amino acid residues at the C-terminal. Aβ can assemble in oligomeric channel structures, as shown in model plasma membranes [87 , 103], a property that would confer cell toxicity. Although Aβ are one of the two hallmarks of AD, their production under normal conditions suggests that they play an important physiological role. For instance, Aβ₄₀ and Aβ₄₂ are present in the cerebrospinal fluid at concentrations of approximately 1,500 pM and 200 pM, respectively, and at concentrations are 60 pM and 20 pM in plasma, respectively [104]. Although Aβ have been considered a harmful byproduct of AβPP processing, they also play a beneficial role in the regulation of memory in humans [105], neurotrophic effect in differentiating neurons [106], neuroprotection, growth and survival in in vitro cultures of rat cortical neurons [107, 108], as well as synaptic plasticity [109]. Of considerable significance (to be discussed below), Aβ peptides may also play a role as antimicrobial agents in the brain. However, the notoriety of Aβ resides in their association with AD. In fact, accumulation of deposits of Aβ in a filamentous (insoluble) form is associated with neuronal degeneration and cortical atrophy [6 , 110]. This finding has served as the basis to the amyloid cascade hypothesis of AD. This hypothesis has been the predominant framework for research in AD since it was initially put forward [111]. In essence, the hypothesis postulates that deposits of Aβ in senile plaques is the cause of AD and “that neurofibrillary tangles, cell loss, vascular damage and dementia follow as a direct result of this deposition”. However, data accumulated over the years have shed doubts on this hypothesis as the major, if not the only cause of AD. The controversies have been the subject of recent reviews arguing against the hypothesis [112] or favoring its modification as an essential component of the complex AD picture [17 , 114]. Currently, it is likely that deposits of Aβ are initiators of a complex pathogenic cascade that involves immune/inflammatory responses [14 , 115–118], tau aggregation [119], neuronal cell death, and neurodegeneration. 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 [120 –123], or both. In addition, the defect in Aβ clearance is further aggravated by age-related immune changes such as immunosenescence and inflamm-aging [124 –128].

INADEQUATE CLEARING OF Aβ PEPTIDES: A COMPONENT OF THE INITIATOR PROCESS THAT LEADS TO AD?

Microglial cells fulfill immunomodulatory functions in the brain and are recognized as resident macrophages [123]. These cells migrate to the site of insult in response to invading pathogens and brain injury [129]. The nature of these injuries may be brain trauma [130], damaged neurons [131], the presence of amyloid plaques, and Aβ aggregates [14]. Microglial cells are involved in Aβ clearance [40, 132]. Therefore, they play a determining role under normal conditions but their activity may be reduced under pathological conditions [14], notably when there has been excess production of Aβ. This situation may contribute to progression to AD and may be part of the onset of the disease. Microglial cells express several plasma membrane receptors for Aβ. These are scavenger receptors, receptor for advanced glycation endproducts, CD36, Fc receptors, and toll-like receptor [133]. Occupation of these receptors induces the switch to a neurotoxic state and the production of the inflammatory cytokines interleukin 1β and 6 (IL-1β, IL-6) and tumor necrosis factor α as well as reactive oxygen species and nitric oxide [14 , 135]. These cytokines are responsible for the neuroinflammation associated with AD [51 , 136–140], including apoptosis and necrosis of damaged neurons [141]. Furthermore, these inflammatory cytokines may negatively influence Aβ clearance, further increasing their accumulation as a result of alteration of Aβ receptor function [142]. In addition, this pathological situation is aggravated by the increased permeability of the blood-brain barrier (BBB) in aging and AD [14 , 144]. Alteration of the BBB allows increased communication between the brain and the periphery, thus establishing a pernicious cycle of sustained immunoinflammatory status. This possibility has been recently reviewed and discussed at length in a recent paper by Goldeck et al. [14]. In essence, initial insult to the brain (trauma, pathogenic infection, Aβ deposits) would trigger activation of microglia and astrocytes and increase the production of Aβ and inflammatory cytokines. On the one hand, the chronic load of Aβ would overwhelm the phagocytic activity of microglia, favoring their deposit as amyloid plaques and the generation of neurofibrillary tangles of hyperphosphorylated tau. On the other hand, the release of brain inflammatory mediators to the periphery through damaged BBB would trigger the peripheral inflammatory response of innate and adaptive immunity. The peripheral inflammatory response in turn would favor the release of pro-inflammatory cytokines and other mediators that would reach the brain through altered BBB. This irreversible pernicious cycle of interconnections between inflammatory processes in the brain and their transmission to the periphery, and back to the brain, would further increase neuronal damages and cripple synaptic communications leading to irreversible progression to AD. This pathological situation may become amplified with age due to immunosenescence/inflamm-aging (Fig. 2).

BRAIN INFECTION BY VIRUSES OF THE HERPESVIRIDAE FAMILY

The case of herpesviridae viruses: Herpes simplex virus-1 (HSV-1) and cytomegalovirus (CMV)

The realization that infectious organisms are involved in the etiology of AD has been gaining momentum in the scientific community. For instance, two editorials signed by several investigators in the field have made convincing arguments in favor of this hypothesis [145, 146]. Various infectious agents have been associated with cognitive decline and the possible onset and progression of AD (Table 1).

A number of reviews have summarized the association between bacteria (particularly Spirochetes), viruses, fungi, and protozoans, and the fact that these agents can be detected in the brain of AD patients, specifically in senile plaques [26 , 148]. With respect to viral infections, it has been suggested that they are a contributing factor to AD [26 , 146]. In fact, the hypothesis of microbial agents as a possible cause of AD dates back to close to 25 years [147 –154]. In 1998, Balin et al. [151] suggested that infection with Chlamydia pneumoniae (now re-named Chlamydophila pneumoniae) was a high risk factor in the development of AD. Furthermore, Itzhaki et al. [46] suggested that infection with HSV-1, when it was present in the brain of carriers of the APOE ɛ4 alleles, was a risk factor for the development of AD. This hypothesis was in agreement with previous suggestions of the involvement of viruses in neurodegeneration [155] and, HSV-1 in AD [156]. HSV-1 is frequently found in amyloid plaques [48 , 158]. In addition, other members of the herpes virus family, namely HSV-2, CMV, and HHV-6, have also been detected in the brain of AD patients [159] or have been associated with its pathogenesis. HSV-1 is a virus, which infects most humans early (before 10 years of age) in life in settings with low socio-economic conditions, including the “developed world” prior to 1970. However, HSV-1 infection is now acquired later, including increased sexual transmission [160]. The virus is able to remain latent during the whole life of the infected individuals [161]. HSV-1 is capable of escaping immune recognition by remaining hidden in the trigeminal ganglions but can be reactivated under conditions of immunodeficiency or stress. Under these conditions, HSV-1 can re-infect the host [162] and colonize the hippocampus and fronto-temporal lobes [154]. Reactivation of HSV-1 can have minor effects but can, in some cases, trigger lethal herpetic encephalitis that also occurs in the same areas of the brain as those affected in AD (hippocampus and frontal and temporal cortical lobes) [163 –165].

In the case of CMV, immunodetection has been used to show its association with AD [166]. However, conclusions of these findings have led to a debate [167, 168] whether the association with AD was supported by sufficient evidence, leaving the question unanswered. Recently, it has been reported that CMV behaves as a cytokine-related promoter of inflammation in relationship to AD [169].

Aβ AS ANTIMICROBIAL PEPTIDES (AMP) AGAINST MICROORGANISMS

Aβ peptides can self-assemble into Aβ structures, a common feature of misfolding for pathological proteins and can form channel structures in cellular plasma membranes [87 , 171], a property that resembles that of channel-forming toxins [172]. Consequently, the formation of leaky channels or pores induces lysis of the targeted organism, leading to cell death. The fact that the cytotoxicity of Aβ was related to its aggregated form and much less or none to its monomer or fibrillar form [173], led to the possibility of a parallel between their channel-forming property and their activity as AMP. This possibility was investigated by Soscia et al. [174]. In this groundbreaking publication, the authors compared the AMP activity of Aβ₄₀ and Aβ₄₂ peptides to that of LL-37, the only human member of the cathelicidin AMP family [175, 176]. Results showed that the Aβ peptides displayed AMP activity against eight of twelve clinically relevant infectious microorganisms (Table 2). Furthermore, colony-forming unit assays revealed that Aβ possessed AMP potency equivalent or even superior to LL-37 and that this activity was reduced by neutralizing antibody depletion. Of significance, the authors reported that Aβ-containing brain homogenates from AD patients displayed AMP activity against Candida albicans, in contrast to homogenates from AD-free subjects. On the basis of Aβ being localized at the membrane of Enterococcus faecalis, for example, the authors suggested that this observation was consistent with the interpretation that Aβ peptides associated with the bacterial membrane. The bulk of these observations led the authors to conclude that, “Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies”.

Aβ AS AMP AGAINST INFECTIOUS VIRUSES

Aβ-dependent inhibition of influenza virus replication

A new picture is slowly emerging with respect to a protective role of Aβ against viral infection. Three recent publications have reported this property in inhibition of replication of influenza [177] and HSV-1 virus [178, 179]. For instance, White et al. [177] have investigated the antiviral effect of Aβ₄₀ and Aβ₄₂ peptides on the replication of seasonal H3N2 and pandemic H1N1 strains of influenza A virus in vitro. Influenza viruses are enveloped RNA viruses that belong to the Orthomyxoviridae family [180, 181]. They are highly contagious and cause acute respiratory distress. Influenza viruses are still the cause of significant morbidity and mortality worldwide. The most severe recorded pandemic occurred in 1918 and has been known as the Spanish flu which caused 40 millions deaths [182]. According to their core protein, influenza viruses are classified into three types, A, B, and C [180, 183]. The influenza A viral particle possesses a lipid envelope, which is derived from the host’s cell membrane during the process of virus budding and three virus-specific envelope-embedded proteins which are haemagglutinin, neuraminidase, and matrix ion channels (Fig. 3). The virus binds to sialic acid-galactose decorated glycoprotein receptors on the surface of respiratory epithelial cells, fuses with the host plasma membrane, is internalized and transfers its genetic material to the nucleus [184, 185].

In the case of White et al.’s work [177], the authors showed that Aβ peptides displayed neutralizing activity when either strain of virus was preincubated with the Aβ peptides in assays of infection of two different epithelial cell lines. Of interest, data showed that the Aβ₄₂ isoform had greater activity than the Aβ₄₀ isoform. Of significance, data suggested that Aβ peptides established interactive bonds with the virus. This interpretation was further confirmed by turbidimetry assays (Fig. 4) and confocal experiments that showed that Aβ induced aggregation of influenza virus. In addition, it was shown that Aβ peptides reduced viral uptake by epithelial cells, increased virus uptake by neutrophils and reduced pro-inflammatory cytokine IL-6 production by these cells. The authors did not provide a definitive mechanism of action of Aβ in these studies but suggested the possibility that Aβ-dependent interference of influenza virus infectivity could be related to alteration of the integrity of the viral envelope,

AMP activity of Aβ peptides against HSV-1 replication in vitro

HSV-1 is a member of the Herpesviridae family of virus that causes lifelong latent infections. It is a double-stranded DNA virus composed of a linear genome. From a structural standpoint, it is composed of an external envelope derived from the nuclear membrane of the host cell that is acquired in the process of virus budding. The envelope is made of a lipid bilayer membrane and decorated with several types of membrane-embedded glycoproteins that protect the encapsidated DNA and its tegument proteins [186] (Fig. 5).

The first step in HSV-1 infection is attachment and fusion of the viral envelope with the cell plasma membrane. Three glycoproteins of the viral envelope play a central role in attachment and infection of target cells [187, 188]. These glycoproteins are glycoprotein B (gB) and heterodimeric glycoprotein H/glycoprotein L (gH/gL). A number of additional viral envelope proteins participate in cognate cellular receptor recognition to ensure viral tropism [189]. This process induces formation of pores, which allow entry of the DNA-containing nucleocapsid into the cytoplasm [189]. gB is a single-pass glycoprotein that comprises 904 amino acid residues that are organized into five regions/domains according to the tridimensional structure of the protein [190]. These are the ectodomain (positions 1–774), the membrane proximal region (MPR), which extends from positions 713 to 763, the transmembrane domain (positions 775–795) and the cytoplasmic domain (positions 796–904).

Bourgade et al. [178, 179] have reported results of investigations designed to answer the question whether Aβ₄₀ and Aβ₄₂ peptides would behave as AMP in in vitro assays of HSV-1 infection of fibroblast, epithelial and neuroglioma cell lines. Data showed that both Aβ isoforms but not scrambled peptides (control), inhibited HSV-1 replication in these cells when added 2 h prior to or concomitantly with virus challenge (Fig. 6). Of significance, Aβ peptides were inefficient when added 2 or 6 h after exposing the cells to the virus. In contrast, comparative experiments using LL-37, a recognized bona fide AMP [176], revealed that the mode of action of Aβ peptides differed dramatically. Of significance, the inhibitory effect of Aβ was not observed when cells were challenged with an adenovirus (hAd5). These observations suggested that 1) Aβ peptides bound to HSV-1, as in the case of Aβ interaction with influenza virus [177], 2) the result of these interactions created interference with the process of viral fusion with the target cells, 3) Aβ interacted with HSV-1, a virus that possesses an envelope but not with envelope-free adenovirus, 4) the AMP effect of Aβ differed from that of LL-37 and, 5) Aβ initiated their anti-viral effect prior to HSV-1 entry into the cells. The bulk of these observations led the authors to conclude that, “Aβ peptides represent a novel class of antimicrobial peptides that protect against neurotropic enveloped virus infections such as HSV-1. Overproduction of Aβ peptide to protect against latent herpes viruses and eventually against other infections, may contribute to amyloid plaque formation, and partially explain why brain infections play a pathogenic role in the progression of the sporadic form of AD.”

The same group sought [179] to obtain additional evidence regarding the protective role of Aβ against HSV-1 infection in in vitro co-cultures of neuroglioma (H4) and glioblastoma (U118-MG) cell lines, a model that allows the study of the mutual influence of these cells in the production/effect of Aβ. Whereas H4 cells produced appreciable levels of HSV-1 upon initial infection, the level of virus production did not increase with continued incubation. Addition of a BACE-1 inhibitor to prevent Aβ production increased HSV-1 production several fold, suggesting an inhibitory effect due to endogenous Aβ generation. Quantification of Aβ₄₂ in HSV-1-infected H4 cells confirmed a robust production of Aβ₄₂ in the supernatant in response to HSV-1 infection. As expected, U118-MG cells produced low levels of Aβ₄₂. However, Aβ₄₂ production in co-cultures was low, suggesting interference due to the presence of glioblastoma cells or uptake of Aβ₄₂ by these cells. Confocal experiments confirmed that glioblastoma cells captured Aβ₄₂, in agreement with what has been reported for microglial cells in Aβ clearance in the brain [40, 132]. Further evidence for the protective effect of Aβ₄₂ against HSV-1 replication was obtained by transfer of supernatants of H4 cells infected with HSV-1 (conditioned supernatants) that were assayed in de novo cultures of H4 cells challenged with HSV-1. It was found that conditioned supernatants conferred protection against HSV-1 infection, likely due to the presence of Aβ because similar supernatants generated by BACE-1 inhibitor-treated H4 cells were ineffective. The protection was not due to the presence of interferon alpha in the supernatants (Fig. 7). The authors concluded, “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”.

PUTATIVE MECHANISMOF Aβ-DEPENDENT INHIBITIONOF HSV-1 INFECTION

There has not been any clear molecular mechanism suggested to explain the AMP activity of Aβ in inhibition of infection by HSV-1. However, Bourgade et al. have suggested that sequence homology between the MPR of the gB fusion protein [191] and Aβ could provide a clue in this matter [178]. In this connection, it has been shown that the MPR serves to temporarily cover or shield lipid-associating moieties or fusion loops of gB [192]. The homology of sequence of Aβ peptides suggests that they could bind to these fusion loops and prevent the fusion process. Overall, the combined results [178, 179] of Bourgade et al. led to the following observations:

Aβ did not enter the cells that are the target of HSV-1, as shown by confocal experiments.

The anti-viral action of Aβ action occurred outside of the target cell.

Aβ loosely interacted with target cells, as shown by washing experiments.

Aβ protection was related to the time-sequence of its addition to cells challenged with HSV-1.

Aβ anti-viral protection was efficient before or concomitant with HSV-1 challenge but not after HSV-1 had time to fuse with target cells.

HSV-1 did not enter Aβ-treated cells.

Aβ protective effect was observed in the case of an enveloped virus but not an envelope-free virus.

Aβ bound to HSV-1, as shown by experiments in a cell-free system and molecular proximity FRET experiments (unpublished).

Sequence homology between the MPR region of gB and Aβ suggested that this region of the fusion protein which is important to maintain protein stability and viral fusion [192] could be an intra target site resulting in interference with viral fusion.

Can the MPR region of fusogenic HSV-1 gB be a target of Aβ?

gB is a conserved protein essential to the cell-entry machinery of herpes viruses. Its MPR region is very hydrophobic and thought to lie in juxtaposition to the plasma membrane, facilitating merger of the HSV-1 envelope. It is also thought to form a pedestal for the trimeric ectodomain of gB [193] and to shield the fusion loops prior to gB triggering of viral fusion [192, 194]. Herpes infection is a multi-stage process that initially involves viral glycoproteins gD, gB, gH, and gL and their binding to the cellular membrane components nectin-1, herpesvirus entry mediator and a modified heparan sulfate [187, 195]. gB MPR region plays a key role in herpes virus association with the target cell and viral fusion and entry. For instance, mutations of non-variant residues in the MPR region markedly decreased infectivity of HSV-1 in in vitro assays [196]. In addition, Hannah et al. [197] have performed a series of mutations (deletion, truncation) in the MPR region and these have revealed that the purified mutant proteins failed to bind to liposomes. These observations led the authors to conclude, “that the ability of the herpes simplex virus (HSV) glycoprotein B (gB) fusion protein to interact with the host membrane is regulated by its membrane-proximal region (MPR), which serves to cover or shield its lipid associating moieties (fusion loops). This in turn prevents the premature binding of gB with host cells and provides a level of regulation to the fusion process”. The bulk of these observations, along with those that presented evidence for a relationship between MPR and the ability of the fusion loops of gB to associate with the plasma membrane [194, 197], provide solid arguments for a regulatory role of MPR in the initial steps of HSV-1 infection. The question thus arises to explain the antiviral effect of Aβ against HSV-1 infectivity. An intriguing possibility to account for the selective effect of Aβ peptides may reside in the fact of the homology of sequence between the MPR and Aβ, as depicted (Fig. 8). Aβ could compete with the MPR region and (partially) disturb the pre-fusion structure of gB, thus altering the spatial arrangement of its fusion loops and inhibiting its action which is essential for HSV-1 fusion [197]. If this hypothesis were valid, it would support the series of observations of Bourgade et al. [178, 179] (outlined above) and provide a working framework for further investigations concerning the mechanism of the antiviral AMP properties of Aβ peptides toward (enveloped) HSV-1. In addition, the mechanism may provide novel avenues in the management of AD. However, Bourgade et al.’s data [178, 179] did not exclude the possibility of a selective alteration of the HSV-1 envelope as a result of pore formation by Aβ that would result in inhibition of HSV-1 infection.

FUTURE DIRECTIONS

The findings that Aβ display antimicrobial [174] and antiviral [177 –179] activity open the way to a novel concept concerning the physiological role of these peptides as protectors against pathogenic aggression of the brain rather than being exclusively associated with cytotoxic components of AD. From a mechanistic standpoint, data are consistent with Aβ interaction with viruses [177, 178]. We propose a minimal model whereby Aβ peptides do not enter the target cells but interfere with HSV-1 infection prior to its fusion with the cell plasma membrane (Fig. 9), thereby inhibiting infection by HSV-1.

However, many questions remain unanswered concerning the AMP property of Aβ peptide:

From the standpoint of the physiological properties of Aβ

Can the observations made in the case of Aβ-dependent interference of HSV-1 infectivity be extended to other members of the Herpesviridae family and to viruses of other families?

Could an analogous induction-for-protection model be evoked for bacterial and, notably spirochete latent infection that would lead to overproduction of Aβ?

Are Aβ peptides involved in this protective process in the brain?

Could vaccination against Herpesviridae viruses early in life protect against development of LOAD, based on the observation that a large proportion of AD patients are infected with HSV [48]?

From the standpoint of future basic research concerning the mechanism of action of Aβ

What is the minimal Aβ sequence required to inhibit HSV-1 infectivity of target cells in functional assays?

What are the critical amino acid residues of the Aβ sequence essential for their AMP property? Analogs of biological interest could be further analyzed for their ability to assemble and to form fibrils [198] using biophysical techniques [199 –201].

Would the MPR sequence inhibit HSV-1 infection?

Does Aβ (or analogs) interference occur in lipid rafts, which are the preferred site of HSV-1 entry into target cells [202]?

Does the binding of Aβ (or analogs) to gB induce conformational changes? This possibility could be addressed using a GFP-labelled gB that can be functionally expressed in infectious HSV-1 [203].

CONCLUSIONS

Accumulation of Aβ results from insults to the brain and this is a contributing factor to the development of AD. Unraveling the mechanism leading to Aβ accumulation and deposit is of crucial importance to design ways and treatments of this devastating disease. Furthermore, the discovery that Aβ may confer an early protective role as AMP to fight various microbial aggressions in the brain, including HSV-1, opens additional avenues in understanding the complex picture of AD. On the one hand, the role of Aβ as AMP has to be taken into account as an essential component of protection of the brain and, therefore, must be reckoned with in the design of medical interventions that are aimed at eliminating Aβ. On the other hand, the harmful effects of Aβ as cytotoxic products of AβPP processing generated under conditions of brain aggression remain a focus point in the design of targeted prevention/treatment of AD. Understanding the mechanisms that lead to the fine regulation of AβPP processing through the non-amyloidogenic and amyloidogenic pathways, as well as the products of AβPP processing, is a timely and pressing challenge that needs to be resolved to tackle the ongoing threat of AD on a worldwide basis. Time is of the essence and this human problem ought to be among the highest priorities of governingauthorities.

NOTE ADDED IN PROOFS

A provocative paper by Kumar et al. [204] has recently extended the original findings of Soscia et al. [174]. This publication provides solid evidence for the role of Aβ as AMP against fungal (Candida albicans) and bacterial (Salmonella enterotica serotype typhimurium) infections in cultured human cell lines and, in transgenic Aβ-expressing nematodes (Caenorhabditis elegans) and mice. The authors used cultures of human brain neuroglioma (H4) cells and chinese hamster ovary (CHO) cells to assess resistance to C. albicans. Results showed that survival of Aβ₄₀- and Aβ₄₂-expressing H4 and CHO cells was significantly increased as compared to wild-type cells. Of significance, it was observed that supernatants of Aβ-expressing cell cultures were able to form fibrils and oligomers that entangled and clumped C. albicans. These observations gave a clue with respect to the mode of action of Aβ peptides as bona fide AMP. In addition, in vivo experiments using C. elegans engineered to express human Aβ₄₂ revealed that these transgenic worms survived three to four more days following infection of the gut with C. albicans or S. typhimurium, compared to wild-type worms that did not express Aβ₄₂. Four-week-old transgenic (5XFAD) mice that constitutively expressed human Aβ at high levels in the brain but that do not show deposits of Aβ and features of neuroinflammation, were infected intracerebally with S. typhimurium. Control animals were non-transgenic wild-type littermates. Results showed rapid seeding and acceleration of Aβ deposits in the brain of 5XFAD mice that colocalized with invading bacteria which became entangled within fibrils of Aβ deposits. Control mice did not show these features. Of significance, survival of transgenic mice was significantly increased with respect to controls. However, both groups of mice succumbed to infection, suggesting that expression of Aβ conferred only partial resistance to bacterial infection in the brain.

The bulk of the data reported in Kumar et al.’s publication led the authors to suggest a model in which soluble Aβ oligomers initially bind to a heparin domain(s) of the microbial cell wall carbohydrates. Propagating Aβ fibrils initially mediate pathogen agglutination, followed by entrapment of the invading microbes. Aβ recognition of the heparin domain likely involves the peptidic sequence XBBXBX (where X is a hydrophobic or uncharged amino acid residue and B is a basic amino acid residue) that is present in Aβ (positions 12–17, VHHQKL) and in human cathelicidin LL37. The authors concluded that, “Our data are consistent with a protective role for Aβ in innate immunity that uses a classic AMP mechanism characterized by reduced microbial adhesion to host cells and agglutination and entrapement of microbes by Aβ fibrils”. Importantly, Kumar et al.’s data lend further support to the notion that Aβ may play a protective role in innate immunity as an additional line of defense against infectious or sterile inflammatory stimuli.

Footnotes

ACKNOWLEDGMENTS

Work in the authors’ laboratories was supported by grants from the 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 ().

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