Potential role of antimicrobial peptides in the early onset of Alzheimer's disease

Alzheimer’s & Dementia - (2014) 1–7

Perspective

Potential role of antimicrobial peptides in the early onset of Alzheimer’s disease Mick M. Wellinga,*, Rob J. A. Nabuursa, Louise van der Weerda,b b

a Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

Abstract

Cerebral aggregation of amyloid-b (Ab) is thought to play a major role in the etiology of Alzheimer’s disease. Environmental influences, including chronic bacterial or viral infections, are thought to alter the permeability of the blood-brain barrier (BBB) and thereby facilitate cerebral colonization by opportunistic pathogens. This may eventually trigger Ab overproduction and aggregation. Host biomolecules that target and combat these pathogens, for instance, antimicrobial peptides (AMPs) such as Ab itself, are an interesting option for the detection and diagnostic follow-up of such cerebral infections. As part of the innate immune system, AMPs are defensive peptides that efficiently penetrate infected cells and tissues beyond many endothelial barriers, most linings, including the BBB, and overall specifically target pathogens. Based on existing literature, we postulate a role for labeled AMPs as a marker to target pathogens that play a role in the aggregation of amyloid in the brain. Ó 2014 The Alzheimer’s Association. All rights reserved.

Keywords:

Infection; Alzheimer’s disease; Amyloid plaques; Antimicrobial peptides; Contrast agents

1. Introduction Based on the existing clinical criteria, Alzheimer’s disease (AD) can only be diagnosed at a late stage of the disease and with a considerable degree of uncertainty. A definitive diagnosis still requires postmortem detection of neurofibrillary tangles and amyloid plaques. Recently, the combination of cerebrospinal fluid biomarkers and nuclear imaging with positron emission tomography tracers fluorodeoxyglucosefluor-18 and Pittsburgh compound B together with structural magnetic resonance imaging has been introduced in clinical settings to play a supporting role in diagnosing AD, as well as to increase our understanding of the disease pathogenesis [1]. Based on the neuropathologic hallmarks and in vivo biomarkers, the AD-induced neurodegeneration is estimated to start several decades before clinical onset and is believed to have reached a plateau at the actual time of clinical presentation [2,3]. The exact underlying pathogenesis

*Corresponding author. Tel.: 131-71-5266099; Fax: 131-715248256. E-mail address: [email protected]

responsible for the amyloid-b (Ab) imbalance, the hyperphosphorylation of tau, and their intimate association remains one of the major unresolved questions regarding AD. Although the amyloid cascade hypothesis is widely known and accepted [2], we still know very little about what triggers plaque formation, let alone about whether their presence is a cause or only a consequence of the disease. Besides the aforementioned amyloid cascade and tau hypothesis, some claim that the dyshomeostasis of cerebral iron plays an intricate and crucial role [4]. Increased cortical accumulation of iron is often found in the presence of amyloid plaques, and mechanistic explanations for the role of iron include neurotoxicity due to the formation of reactive oxygen species or a dysregulation of myelin maintenance. Both processes could be viewed as positive forward mechanisms as amyloid precursor protein and Ab, tau, and demyelination in turn may contribute to the iron dyshomeostasis [4]. It is clear that as yet no clear single explanation regarding the pathogenesis of AD has been found. In 2010, a hypothesis was postulated that brain infections with bacteria or viruses may play an initiating role in amyloid plaque formation and the development of AD [2]. Our

1552-5260/$ - see front matter Ó 2014 The Alzheimer’s Association. All rights reserved. http://dx.doi.org/10.1016/j.jalz.2013.12.020

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perspective aims to provide evidence for the effect of chronic infections in the development of AD based on existing literature. Second, we propose a potentially novel role for antimicrobial peptides (AMPs) in AD pathology as pathogen-targeting agents for the detection and follow-up of these brain infections with respect to AD. 2. Chronic infections as an initial event in AD pathogenesis As suggested by others, chronic systemic infections may play a crucial role in the initial pathogenesis of AD. Many inflammatory markers are indeed found to be significantly increased in AD subjects, as highlighted in a recent review by Zotova et al. [5]. Apart from chronic infection, vitamin D deficiency, obesity, rheumatism, depression, stress, or type 2 diabetes are proposed as risk factors [6–8]. These factors are known to contribute to the downregulation of the innate immune response [9] and thus increase the risk for a bacterial infection [10] (Fig. 1). These infections lead to persistent inflammatory stimuli, regulated by cytokines that are known to induce stress and alter the immune response [11]. As these stimuli have a detrimental effect on the integrity of the blood-brain barrier (BBB) [12], they provide an opportunity for other pathogens to enter the central nervous system (CNS). Second, the inflammatory response to a systemic infection may indirectly lead to an upregulation of Ab production, thereby initiating the amyloid cascade (Fig. 2). 3. The infiltration of pathogens into the brain As said earlier, inflammatory stimuli may compromise the endothelial layer. Supportive for this hypothesis is the observation that for both sepsis and bacterial meningitis, the BBB is compromised because of the breakdown of intercellular tight junctions caused by the endotoxin lipopolysaccharide and peptidoglycans, particularly within the venules [13]. Moreover, chronic infections alter the integrity of the BBB and may promote the transmigration of monocytes and autoreactive T cells over the brain epithelium. As these cells are potentially infected by pathogens, monocytes and T cells may well be the vehicle to transport bacteria into the CNS [14]. Indeed, persistent subclinical CNS infections have been reported for AD patients, caused by various pathogens such as Chlamydia, Borrelia spirochetes, Helicobacter pylori, herpes simplex virus, and infections related to human immunodeficiency virus that causes acquired immunodeficiency syndrome [14,15]. A complete overview of recent studies regarding the incidence and role of bacterial or viral CNS pathogens in AD pathology is summarized in Table 1. In many cases, the presence of bacterial infections in the brain was confirmed by polymerase chain reaction techniques after autopsy. Therefore, a critical remark must be

made when discussing the causality of AD based only on the results of postmortem studies because from these studies, it remains unclear whether the entry of these pathogens occurred at the onset of AD or is merely the consequence of a leaky BBB caused by inflammatory processes induced by other chronic infections or secondary to AD itself [16]. Unfortunately, currently, no methods are available for in vivo detection of infectious agents in the brain, apart from an invasive brain biopsy. Specific infection imaging agents that target the CNS would be essential to further the knowledge of the causality of these mechanisms. 4. Linking chronic infections to AD onset The (repetitive) infiltration of pathogens in the brain is thought to induce AD directly through CNS (re)infection and the resulting neuroinflammatory response, although no specific bacterial or viral pathogens have been linked conclusively to late-onset AD in humans [17]. Clinical evidence for the role of infections stems from studies on the relation between periodontitis and AD, showing that the presence of serum antibodies to periodontal bacteria associates with an AD diagnosis and even presents an independent risk factor for the future development of AD [18]. Rather than AD being the result of a single pathogen, the diverse bacterial and viral pathogens associated with the disease evoke a similar neuroinflammatory response, thereby initiating the formation of fibrillar amyloid, as will be explained in the next paragraph. Once initiated, the amyloid formation then proceeds through a self-assembling process, which may be accelerated by the neuroinflammatory status of the patient, thus creating a positive feed-forward loop. In line with our hypothesis, recently, a number of animal studies have been published showing that several pathogens (Chlamydia pneumoniae, herpes simplex, Escherichia coli, or Cryptococcus neoformans) play a significant role in the development of amyloid plaque formation [19–22]. These types of models will be of great value to establish the role of bacterial and viral infections in the onset of AD, as well as to test potential imaging markers aiming to detect living and virulent pathogens in the brains. In this respect, AMPs that were developed as specific tracers for imaging infections may play an important role in this [23]. 5. The role of AMPs in AD AMPs are part of the innate immune response and can be found in all living species. In humans, AMPs are produced by phagocytes, epithelial cells, endothelial cells, and many other cell types [24]. They can be expressed constitutively or be induced during inflammation or a microbial challenge. AMPs usually contain less than 50 amino acids and can be classified into three main structural categories: (1) linear peptides adopting an amphipathic a-helical structure such as cecropin, magainin, bee melittin, and human ubiquicidin and histatins; (2) peptides with 1 to 4 disulphide bridges

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Fig. 1. Role of infections and inflammatory pathways in the pathophysiology of Alzheimer’s disease (AD) of the human brains. The sequence of pathogenic events leading to neuronal damage is based on age-related disorders that may impair the host defense system or cause chronic inflammatory responses, which in turn initiate AD pathology. Additional modulators such as atherosclerosis, lipid homeostasis, stress, LPS, and other pyrogens influence the extent of chronic infection in the brain. AMP, antimicrobial peptide; LPS, lipopolysaccharide; BBB, blood-brain barrier; CSF, cerebrospinal fluid; PNS, peripheral nervous system; VLDL, very-low-density lipoprotein; LDL, low-density lipoprotein; APP, amyloid precursor protein LPR-1, lipoprotein receptor-1.

adopting a loop or a b-sheet structure such as defensins; and (3) peptides that are particularly rich in one amino acid (besides cationic amino acids) such as the proline-arginine–rich peptide PR39 of pig neutrophils or the tryptophan-rich indolicidin of bovine neutrophils. Despite the large variety in sequence and molecular conformation, virtually all AMPs have a net positive charge because of an excess of basic residues and approximately 50% hydrophobic amino acids. AMPs have been studied for many decades for their action against pathogens to develop a new generation of antibiotics that prevent the occurrence of drug resistance. Some of them have found their way into clinical trials because of their diagnostic or therapeutic potential [25]. AMPs have different ways of antimicrobial actions, all of which include an interaction with the microorganism membrane. In general, after passing endothelial barriers to penetrate infected tissues, the positively charged AMPs bind electrostatically to negatively charged groups or oxidized lipids located on the outer membranes of pathogens, leading to pore formation with detergent-like action or destabiliza-

tion of the membrane [26–28]. Interactions with membranes of host cells are to a large extent restricted because of protection of the charged groups by membrane lipids and cholesterol, though mitochondrial membranes are vulnerable because of their close resemblance to prokaryotic membranes. There are also a few examples in which membrane domain recognition by AMPs is the leading mechanism. Similar to b-sheeted AMPs, they cause formation of membrane deposits by surface selfaggregation, which leads to membrane destabilization and causes cell leakage [29]. AMPs also trigger immunostimulatory effects including the upregulation of tumor necrosis factor-a and interleukin-8, neutralization of lipopolysaccharide, promotion of wound healing, or display of synergistic interactions with other host defense compounds in various tissues including the brain [30]. The microbicidal activity of moderate levels of AMPs in the brains of AD patients is thought to act through synergistic pathways (Fig. 2), with oligomerization playing a key role in the targeting and permeabilization of membranes

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Fig. 2. Pathways of infiltration of pathogens into the brains, the host response, and the effect of amyloid plaques on immunologic function, including antimicrobial peptides (AMPs) in the human brain. Closed arrows indicate stimulating pathway, dotted arrows indicate suppressive pathway, and red arrows indicate antimicrobial activity.

[29,31]. The AMP microcin E492, naturally produced by Klebsiella pneumoniae to kill competitor bacteria by forming pores in the cytoplasmic membrane, is an example of such a self-assembling peptide. Remarkably, it aggregates in vitro into amyloid-like fibrils very similar to the aggregates observed in AD [32]. The finding that microcin E492 naturally exists, both as functional toxic pores and as harmless fibrils, suggests that protein assembly into amyloid fibrils is an evolutionarily conserved property that can be successfully used by organisms to regulate the aggregation state of these proteins. 6. Ab as an antimicrobial peptide Ab1-42 itself is an underrecognized AMP that has a function in the cerebral innate immune system [31]. In general, soluble monomeric AMPs such as Ab1-42 show little antimicrobial activity, but once they aggregate, the formed b-sheet structures may assemble into antimicrobial pore-forming structures [33]. In vivo evidence for the antimicrobial activity of Ab1-42 against clinically relevant pathogens has been found using immunocompetent b-secretase knockout mice. These mice generate low levels of Ab1-42, resulting in an

increased susceptibility to infections [34]. In addition, a clinical trial with the Ab1-42-lowering agent tarenflurbil led to a significantly increased rate of infections in treated patients with mild or moderate forms of AD [35]. The normal production of other AMPs such as neuro–antimicrobial peptide may be reduced because of a number of factors such as a weakened immune system, damaged defense cells (by either intracellular infections or apoptotic processes), or structural vitamin D deficiency [6]. In those cases, the antimicrobial role is partly taken over by Ab1-42 mediated as a stress response [28,30,36]. Summarizing, acute or chronic systemic infections stimulate the innate immune system in the brain, which increases Ab1-42 production, leading to self-aggregation as an antimicrobial response. These aggregates themselves are also harmful for the host cells. Both the bacterial infection and the formed Ab aggregates result in CNS inflammation, including microglial activation, which in turn again promotes Ab production. 7. AMPs as a marker for infections To test our hypothesis regarding the initiating role of AMPs in AD pathology, it is of importance to follow the

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Table 1 Role of pathogens in CNS infections in the development of Alzheimer’s disease (AD) Pathogen Herpes simplex virus type 1 (HSV1)

Incidence

Mechanisms of infection and AD

 65%–90% of the elderly population harbors the virus.  High risk factor for developing AD for APOE ε4 allele carriers

 De novo infection via the olfactory route.  Via LDLR receptors.  Reactivation (stress, immunosuppression, and other infections).  Increased tau phosphorylation.

 About 90% positive in late-onset AD

 Intracellular infection in microglia, astrocytes, perivascular macrophages, and monocytes.  Stimulates inflammatory response and deposition of amyloid.

 HIV-associated dementia in 40% of the patients.  Increased risk after incomplete response to highly active antiretroviral therapy. Rare

 Inhibition of neprilysin by Tat, an Ab-degrading enzyme.

[14,16,46]

Chlamydia pneumoniae

Human immunodeficiency virus (HIV)

Borrelia burgdorferi

[19,47,48]

[49,50]

 Altered cerebral amyloid metabolism in patients with Lyme neuroborreliosis (LNB).  Effects on tau phosphorylation and levels of APP and P-tau. Toxoplasmosis gondii

30% of the general population  Overproduction of IFN-g and other proinflammatory cytokines, associated with persistent neuroinflammation.

Helicobacter pylori

88% of AD and 89% of MCI patients  Chronic gastritis can lead to malabsorption of vitamins (B-12) and folate.  Elevated Hcy triggers endothelial oxidative damage and AD development.

Escherichia coli

References

Rare  Penetration of the BBB via the host cytosolic phospholipase A2a pathway.  The formation of the extracellular fibers by E coli can be the onset of amyloid formation.

[51,52]

[53] [54,55]

[21,56]

Abbreviations: CNS, central nervous system; APOE, apolipoprotein E; LDLR, low-density lipoprotein receptor; Ab, amyloid-b; APP, amyloid precursor protein; IFN-g, interferon-g; MCI, mild cognitive impairment; Hcy, homocysteine; BBB, blood-brain barrier.

earliest events of the disease, including the initial pathologic changes due to inflammatory reactions in the small blood vessels [37] and the presence of pathogens in the endothelial layer, behind the BBB, or inside neurologic cells. Tracers that can diagnose specific infectious processes and monitor effectiveness of (antimicrobial or anti-inflammatory) therapy are therefore of great promise. During the last decade, various research groups studied the potency of imaging and treatment of drug-resistant pathogens using radiolabeled antimicrobial compounds [24], and the radiopharmacy and pharmacology of cationic AMPs are well established [25]. Examples of radiolabeled natural AMPs, which have been intensively evaluated for the imaging of infections in animal models, are (recombinant) lactoferrins, defensins, histatins, and ubiquicidin [23,38,39]. These studies demonstrated that radiolabeled AMPs pass the vascular endothelium efficiently and are able to detect various types of infections (soft tissues, bone, sepsis, and endocarditis) with high sensitivity and specificity [25]. Several clinical trials have been initiated to demonstrate the effectiveness of radiola-

beled AMPs in detecting infections in patients, and up to now, no adverse side effects have been observed [40]. 8. AMPs as potential imaging biomarkers for AD Imaging infections in the brain is problematic as currently available markers cannot pass endothelial linings or the BBB or do not discriminate between infections by pathogens and other neuroinflammatory processes [25]. Currently, no methods are available for in vivo detection of infectious agents in the brain, apart from an invasive brain biopsy. What make AMPs unique and promising is that they pass biological barriers and penetrate infected tissues. This is particularly true for the small synthetic derivatives under 500 D, which fulfill most of the rule of five given by Lipinski et al. [41] stating that BBB-crossing molecules should be relatively small and be moderately lipophilic to ensure transport through phospholipid-based barriers, including the BBB. Although AMPs have hardly been studied in the context of the brain, some studies do indicate that AMPs,

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including Ab, are able to cross the BBB. The exact mechanism seems to be variable and, dependent on the AMP species, may be through passive diffusion, receptor-mediated uptake, or through self-assembly into cationic nanoparticles [42–44]. One of the most promising AMPs for BBB delivery may be lactoferrin as this AMP has a specific transport system to the brain, mediated by an endocytotic mechanism involving high- and low-affinity binding sites [45]. Lactoferrin has not only been detected in different areas in the brain after systemic or oral administration but is also intrinsically upregulated in the brain during infectious processes [45]. 9. Conclusion The pathology of AD is still only partially understood, and clinical trials with amyloid-targeting drugs in humans are so far unsuccessful. There are indications that besides the amyloid cascade hypothesis, there may be other initiators of the AD pathogenesis. In this perspective, we addressed the potential role of chronic infections in the pathogenesis of AD and highlighted the potential role of AMPs. We discussed the role of Ab as an AMP in this respect, posing the hypothesis that Ab (over)production is the result of an immune response to a brain infection. Second, AMPs in general readily pass endothelial linings to penetrate infected tissues and target specific pathogens, which make them good candidates as ligands to image CNS infections. Future research into the role of AMPs in AD patients, either as an innate part of the immune response or as imaging ligands, will be dependent on the technical ability to detect brain infections in vivo. In vivo methods will be essential to prove or disprove the hypothesis that brain pathogens play an initiating role in the development of AD. Acknowledgments The authors thank Gerrit Kracht for preparing the artwork and figures.

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