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. Author manuscript; available in PMC: 2018 Aug 3.
Published in final edited form as: Neuron. 2018 Jul 11;99(1):56–63.e3. doi: 10.1016/j.neuron.2018.06.030

Alzheimer’s Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection

William A Eimer 1,2, Deepak Kumar Vijaya Kumar 1,2, Nanda Kumar N Shanmugam 1,2, Alex S Rodriguez 1,2, Teryn Mitchell 1,2, Kevin J Washicosky 1,2, Bence György 2, Xandra O Breakefield 2, Rudolph E Tanzi 1,2,*, Robert D Moir 1,2,3,*
PMCID: PMC6075814  NIHMSID: NIHMS982565  PMID: 30001512

SUMMARY

Amyloid-β peptide (Aβ) fibrilization and deposition as β-amyloid are hallmarks of Alzheimer’s disease (AD) pathology. We recently reported Aβ is an innate immune protein that protects against fungal and bacterial infections. Fibrilization pathways mediate Aβ antimicrobial activities. Thus, infection can seed and dramatically accelerate β-amyloid deposition. Here, we show Aβ oligomers bind herpesvirus surface glycoproteins, accelerating β-amyloid deposition and leading to protective viral entrapment activity in 5XFAD mouse and 3D human neural cell culture infection models against neurotropic herpes simplex virus 1 (HSV1) and human herpesvirus 6A and B. Herpesviridae are linked to AD, but it has been unclear how viruses may induce β-amyloidosis in brain. These data support the notion that Aβ might play a protective role in CNS innate immunity, and suggest an AD etiological mechanism in which herpesviridae infection may directly promote Aβ amyloidosis.

In Brief

Eimer et al. report that Aβ traps herpes viruses in insoluble deposits called amyloid. High amyloid accumulation is known to drive Alzheimer’s disease pathology. Hence, this study suggests that active herpes infections in brain may accelerate amyloid deposition and the progression of Alzheimer’s disease.

INTRODUCTION

Deposition of amyloid-β peptide (Aβ) as β-amyloid plaques is a hallmark pathology of Alzheimer’s disease (AD). Traditionally, Aβ has been characterized as a functionless catabolic byproduct and pathways leading to β-amyloid generation as intrinsically pathological. However, mounting data suggest this long-standing model for Aβ pathogenesis requires revision. Recent findings have identified Aβ as an antimicrobial peptide (AMP), and suggest Aβ deposition may be a protective innate immune response to infection (Bourgade et al., 2015; Bourgade et al., 2016; Kumar et al., 2016; Soscia et al., 2010; White et al., 2014). A physiological role for Aβ as an AMP is also consistent with the peptide’s remarkably high sequence conservation across multiple phyla - the human Aβ sequence is at least 400 million years old and is found in 60–70 % of vertebrate species (Luna et al., 2013). We have proposed that in AD, normally protective antimicrobial pathways mediated by Aβ oligomerization are over activated, either by real or incorrectly perceived infection. Ongoing Aβ deposition drives neuroinflammation, leading to neuropathology and widespread neuronal death. We call this model the antimicrobial protection hypothesis of AD.

AMPs are evolutionarily conserved proteins that mediate a diverse range of protective functions in innate immunity, including antibiotic-like microbial killing and immuno-modulating activities. Oligomerization is integral to normal AMP function, and soluble oligomers are the bioactive species in a range of innate immune pathways (Arnusch et al., 2007; Chu et al., 2012; Hoover et al., 2000; Oren et al., 1999; Xhindoli et al., 2014). Amyloid fibril generation is also important for AMP activities, mediating disruption of microbial cell membranes (Sood et al., 2008), neutralization of bacterial endotoxins (Wang et al., 2014), and pathogen agglutination and entrapment (Chu et al., 2012; Torrent et al., 2012). However, dysregulated AMP oligomerization can also lead to serious pathologies, including inflammation, tissue degeneration (Paulsen et al., 2002; Pereira et al., 1996; Reinholz et al., 2012; Scarpa et al., 2012), and deposition as amyloid in at least three human amyloidopathies (Araki-Sasaki et al., 2005; Ciornei et al., 2006; Kee et al., 2008; Linke et al., 2005; Millucci et al., 2011; Pálffy et al., 2009; Reinholz et al., 2012; Scarpa et al., 2012; Yamaguchi et al., 2007; Zhao et al., 2008). Thus, AMP oligomerization carries a potential for protective/damaging duality in human health.

Alois Alzheimer first proposed infection may play a role in AD (Fischer, 1907). However, until recently there has been limited support for an infection-based AD etiology (Itzhaki et al., 2016). Renewed interest in possible roles for infection has been driven, in part, by recent reports of elevated levels of pathogen-derived epitopes in AD brain (Alonso et al., 2014; Harris and Harris, 2015; Pisa et al., 2015) and emerging experimental, genetic, and epidemiological data suggesting innate immune mediated inflammation propagates neurodegeneration (Griciuc et al., 2013; Heneka et al., 2015; Perez-Nievas et al., 2013). Herpes simplex virus 1 (HSV1) is the human pathogen most frequently linked to AD amyloidosis (Harris and Harris, 2015; Itzhaki, 2014; Itzhaki et al., 1997; Mawanda and Wallace, 2013). Herpesviridae proteins are reported to interact with the products of many AD susceptibility genes (Carter, 2008). HSV1 upregulates Aβ generation (Wozniak et al., 2007) and herpesviridae DNA signals co-localize with amyloid plaques in AD brain (Wozniak et al., 2009). Here, we investigated the activities of Aβ against the neurotropic herpesviridae viruses HSV1 and herpesvirus 6A (HHV6A) and 6B (HHV6B). Herpesviridae are ubiquitous human pathogens with neuroinfection in the general population approaching 100% (Campadelli-Fiume et al., 1999; Jamieson et al., 1991). HSV1 undergoes acute and episodic reactivation between long periods of dormancy. The continuous low-level replication of HHV6A and HHV6B is implicated in an increasing spectrum of chronic inflammatory diseases with foci both inside and outside the CNS (Hill and Venna, 2014). In this same issue, an increased abundance of Roseoloviruses HHV6 and HHV7, and HSV1 has also been reported in the brains of AD patients (Readhead et al., 2014).

In this study, we report that Aβ oligomers inhibit HSV1 infection in vitro and significantly protect 5XFAD transgenic mice from acute viral encephalitis. Furthermore, we demonstrate that Aβ binds and agglutinates HSV1 and HHV6 viruses, and identify herpesviridae envelope glycoproteins as binding targets. Our data are consistent with rapid and dramatic viral seeding of β-amyloid in the brain, in which various conformers of Aβ serve as innate intra-CNS mediators of resistance that protects the brain against herpesviridae infection.

RESULTS

Aβ-mediated antiviral activity was tested in mouse encephalitis and cell culture infection models. For the encephalitis model, transgenic AD mice (5XFAD) and wild-type (WT) littermates were compared for survival following stereotactic injection of 5×106 PFU/μl of HSV1 into the hippocampal region of each brain hemisphere. Mice are poor experimental models for investigating HHV6 infections (Lusso, 1996). The human cell receptors for HHV6A (CD46) and HHV6B (CD134) have low sequence homology with murine homologues. Thus, HHV6 infectivity was investigated in cell culture, including human neuroglioma (H4) monolayer models and the recently reported three-dimensional (3D) human stem cell-derived neural cell culture system that recapitulates both β-amyloid plaque and neurofibrillary tangles (Choi et al., 2014). Oligomer-depleted synthetic Aβ were prepared as previously described (Kumar et al., 2016). Cell-derived oligomeric Aβ species were harvested from conditioned media generated by H4 cells stably transformed with a BRI-Aβ42 fusion protein (H4-Aβ42) or a Chinese hamster ovary cell line over-expressing the amyloid β-protein precursor (APP) and β-secretase (CHO-CAB) (Kumar et al., 2016). CHO-CAB cells express both 40 (Aβ40) and 42 (Aβ42) residue Aβ isoforms while H4-Aβ42 cells predominately express Aβ42. Conditioned H4-Aβ42 and CHO-CAB media predominately contain polydisperse polymorphic soluble oligomer species assembled from between 2 and 20 Aβ monomer unit (Kumar et al., 2016). Synthetic Aβ peptides used in experiments were prepared under conditions that minimize soluble oligomer generation.

Aβ protects against HSV1 encephalitis in 5XFAD transgenic mice.

To interrogate the protective antiherpetic activity of elevated Aβ expression we first employed a mouse HSV1 encephalitis mouse model. Five-to-six-week old 5XFAD transgenic mice constitutively express human Aβ in the brain at high levels, but exhibit neither the β-amyloid deposits nor features of neuroinflammation observed in older (> 4 month old) animals (Oakley et al., 2006). 5XFAD mice (N = 14) and WT (N = 11) littermates were administered HSV1 by intracerebral injection and followed for survival. Infected 5XFAD mice expressing Aβ showed significantly (P=0.045) increased survival compared to WT non-transgenic littermates (Figure 1A). Consistent with increased HSV1 resistance, weight loss was also significantly less (P=0.013) for 5XFAD mice compared to WT littermates at 24 hrs post-infection (Figure 1B). Sham viral injections did not result in 5XFAD or WT mouse mortality. Data are consistent with protective antiherpetic activity mediated by human Aβ expression in transgenic 5XFAD mice.

Figure 1. Aβ 42 expression increases host survival in a HSV1 encephalitis 5XFAD mouse model.

Figure 1.

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Transgenic mice (5XFAD) expressing human Aβ were compared against wild type (WT) littermates for survival following bilateral intracranial injections of HSV1. Following injection of viable HSV1, WT and 5XFAD mice were followed for (a) survival and (b) weight loss. No mortality was observed among sham-infected control. Statistical significance was calculated by log-rank (Mantel-Cox) test for survival and statistical means compared by t-test for weight loss. Survival analysis data were pooled from five independent experiments.

Aβ inhibits HSV1 infection of host cells in culture.

The protective activities of Aβ against HSV1 were further characterized in a H4-N monolayer infection model using red fluorescent protein (RFP) labeled virus. Compared to naïve control samples (H4-N and CHO-N), host cell-associated HSV1-RFP signal was significantly attenuated with coincubation in transformed H4-Aβ42 and CHO-CAB conditioned media (Figure 2). Consistent with Aβ-specific antiviral activity, anti-Aβ immunodepleted (ID) H4-Aβ42 and CHO-CAB media did not inhibit HSV1. HSV1 was also not inhibited by pre-incubation of host cell with Aβ or addition of peptide following infection (Figure S1A). Protection was concentration-dependent, and consistent with our previous findings (Kumar et al., 2016) for fungal and bacterial pathogens, cell-derived Aβ oligomers inhibit HSV1 at low nanomolar concentrations while synthetic peptide treated to disaggregate assemblies require micromolar concentrations (Sup. Figure S1B).

Figure 2. Cell-derived Aβ 42 oligomers mediate HSV1 resistance in cell culture monolayer infection models.

Figure 2.

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Confluent human H4-N cell monolayers were co-incubated (2 hrs) with HSV1-RFP fusion virus and conditioned media from transformed H4-Aβ42 (H4-Aβ42) or CHO-Aβ (CHO-Aβ) cells, anti-Aβ immunodepleted (ID) H4-Aβ42 (ID H4-Aβ42) and CHO-Aβ (ID CHO-Aβ) media, and synthetic (Aβ42) or scrambled (scAβ42) peptides treated to disrupt soluble oligomeric species. Host cell HSV1-RDP infection was assayed by flow cytometry. Signal shown as percentage of naïve H4-N, CHO-N, or unconditioned media for H4-Aβ42, CHO-Aβ, and Aβ peptides, respectively. Bars are mean signal of replicates (n=6) ± SEM. Statistical mean comparisons of naïve and transformed signal were by t-test. Flow cytometry gating strategy shown in supplemental figure 7. Data are representative of at least 6 independent experiments for each condition.

Aβ carbohydrate binding mediates targeting of herpesviruses

Next, we investigated the pathway mediating targeting of herpesviruses by Aβ. Previously, we showed Aβ inhibits bacterial and fungal pathogens by binding directly to microbial surface carbohydrates (Kumar et al., 2016). Aβ binding of microbial cells is mediated by the peptide’s heparin-binding domain. Aβ oligomers, but not monomeric peptide, show heparin-binding activity (Kumar et al., 2016; Watson et al., 1997). Thus, soluble oligomers appear to be the Aβ species targeting and binding microbes. Carbohydrate binding promotes Aβ fibrillization and leads to protofibril generation on microbial surfaces. Bound protofibrils first inhibit host cell adhesion by pathogens. Then, propagating Aβ fibrils mediate agglutination and sequestration of microbes within fibrillar β-amyloid.

The heparin-binding domain of Aβ has previously been shown to target microbial surface carbohydrates in in vitro binding assays (Kumar et al., 2016). We assayed Aβ binding to HSV1 and HHV6 A and B viral particles using a modified Aβ binding-ELISA. Synthetic or cell-derived Aβ was incubated with heat-immobilized virus in microtiter plate wells. β-amyloid generated on viral surfaces was then detected immunochemically with anti-Aβ antibodies. Consistent with Aβ targeting of herpes viruses, binding signal was observed following incubation of synthetic Aβ42 and H4-Aβ42 and CHO-CAB conditioned media with immobilized HSV1, HHV6A, and HHV6B virus (Figure 3A and S2). Cell-derived Aβ oligomers bound herpes viruses at peptide levels reported for normal brain (0.5–2 ng/ml). Consistent with specificity for Aβ binding, signal was attenuated for anti-Aβ immunodepleted culture samples and scrambled Aβ42 peptide. Competitive inhibition of pathogen binding by soluble microbial sugars is a hallmark for AMPs with activities mediated by lectin-like carbohydrate binding (Tsai et al., 2011,b). Consistent with carbohydrate-mediated virus binding, the yeast polysaccharide mannan inhibited Aβ binding to immobilized HSV1 (Figure 3A). Next, we investigated possible carbohydrate moieties on herpesviridae that Aβ may target. Classical heparin-binding AMPs target viral glycoproteins, including LL-37 (Pachon-Ibanez et al., 2017) and θ and α -defensins (Hazrati et al., 2006). Immobilized HSV1 was pre-incubated with antibodies to herpesviridae glycoproteins B (gB), C (gC), D (gD), E (gE), H (gH), or G (gG) prior to incubation with conditioned CHO-CAB media. Compared to untreated wells, signal was attenuated following pre-incubation with anti-glycoprotein antibodies (Figure 3B). Collectively, data are consistent with antiherpetic activities mediated by Aβ binding herpesviridae glycoproteins.

Figure 3. Aβ heparin-binding activity mediates binding of herpesviridae glycoproteins.

Figure 3.

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Aβ binding to heat-immobilized intact whole HSV1 particles was characterized in an Aβ-binding ELISA. (a) Wells containing immobilized HSV1 were incubated with transformed cell conditioned media (CHO-CAB) or synthetic Aβ42 (12.5 μg/ml) peptide (Syn Aβ42) alone or with mannan (+Mannan), or negative control anti-Aβ immunodepleted media (CHO-CAB ID), and probed for anti-Aβ signal. (b) HSV1 wells were pre-incubated with antibodies against HSV1 surface glycoproteins B (α-gB), C (α-gC), D (α-gD), E (α-gE), G (α-gG), and H (α-gH) then incubated with H4-Aβ42 conditioned media. Signal shown as percentage of naïve CHO-N or unconditioned media for CHO-CAB and Aβ peptides, respectively. Bars are mean signal of replicates (n=6) ± SEM. Statistical mean comparisons were done by t-test. Panels show data representative of finding from at least 8 independent experiments.

Soluble microbial sugars inhibited binding signal for CHO-CAB and H4-Aβ42 conditioned media by 56% and 73%, respectively (Figure 3A). However, higher mannan concentrations or other microbial sugars did not further reduce binding under our experimental conditions (data not shown). This suggests Aβ binding of viral glycoproteins may not be the sole pathway mediating targeting of herpesviridae. Consistent with alternative/complementary herpes targeting pathways, Bourgade et al., 2016 report HSV1 inhibition mediated by binding of a proximal region of gB with high sequence homology to Aβ (Bourgade et al., 2016).

Oligomerization of viral bound Aβ inhibits infectivity of herpesviruses

Anti-adhesion and agglutination activities against microbial cells is mediated by Aβ fibrillization following binding of oligomeric species to surface carbohydrates (Kumar et al., 2016). We next investigated whether Aβ fibrillization also mediates the peptide’s ability to inhibit HSV1 and HHV6. Consistent with a fibrillization-mediated inhibition model, transmission electron micrographs (TEM) of HSV1, HHV6A, or HHV6B incubated with H4-Aβ42 media revealed fibrillar structures attached to viral envelopes (Figure 4A). Fibril generation on viral surfaces was rapid, occurring in <15 min. Virus particles were linked by a fibrillar network following 60 min incubation and sequestered within insoluble agglutinates within 2 hrs. Identity of the entrapping fibrillar structures as Aβ42 fibrils was confirmed by anti-Aβ (mAb 4G8) labeling using immunogold nanoparticles (Figure S3A). Incubation of viruses with anti-Aβ immunodepleted control media did not generate fibril networks or lead to agglutination. Data from our 3D neural cell culture system were also consistent with findings from monolayer cell culture models. Under sterile conditions, observable Aβ deposits are normally not generated in the 3D cell neural culture system until the sixth week of culture (Choi et al., 2014). However, extensive Aβ positive deposits were observed in three-week old cultures within 48 hrs of infection with HSV1, HHV6A, or HHV6B (Figure 4B and 4C). Following staining with Thioflavin S, matrigel Aβ/HSV1 deposits displayed the enhanced fluorescence that mark the presence of amyloid fibrils (Figure S3B-C). Moreover, viral signal co-localized with Aβ deposits generated in cell culture, consistent with viral agglutination and entrapment mediated by fibrillization. In contrast, Aβ aggregates were not observed in control cell cultures infected with UV-inactivated virus (Figure S3D).

Figure 4. Aβ fibrilization mediates viral entrapment in 3D human stem cell-derived neural cell culture and 5XFAD mouse infection models.

Figure 4.

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Aβ fibrillization with HSV1 (HSV1), HHV6A (HHV6A), or HHV6B (HHV6B) infection was characterized in 3D cultures of GFP-expressing human neurons (host cells) and AD mouse models. Cell cultures and mouse brain sections were immunoprobed with anti-HSV1 (α-HSV1), anti-gB (α-gB), or anti-Aβ (α-Aβ) fluorophore labeled antibodies, and analyzed for green (GFP), red (RFP), and blue (405) fluorescence by confocal microscopy. (a) Virus preparations were incubated for 15, 30 or 120 min in H4-Aβ42 conditioned media and analyzed by TEM. (b and c) Three-week old 3D cell cultures were left untreated (Uninfected) or incubated (48 hrs) with virus and fluorescence signals captured under (b) low or (c) high magnification. (d and e) Wild-type (WT) and transgenic AD (5XFAD) mice were injected with HSV1 (+HSV1) or sterile vehicle (Uninfected) and brain sections immunoprobed for HSV1 and Aβ and stained for β-amyloid (β-amyloid) with Thioflavin S (ThS). Panels show (d) wide-field signal from brain 72 hrs following injection with high (lethal) HSV1 titer and (e) high magnification image of Aβ deposits in 5XFAD brain 21 days post-administration of non-lethal viral dose. Panels are representative of multiple image fields (>20) captured for each experimental condition.

The data from the cell culture experiments are consistent with antiherpetic Aβ activities mediated by β-amyloid generation. Consistent with this model, protective Aβ entrapment of infecting bacteria induced rapid seeding of β-amyloid in 5XFAD brain (Kumar et al., 2016). Thus, we next tested whether HSV1 infection also seeds Aβ deposition in young 5XFAD mice. Young 5XFAD mice do not develop Aβ deposits until 10–12 weeks of age. The brains of five-to-six-week old 5XFAD mice and WT littermates given intracranial HSV1 injections were analyzed for Aβ deposition. Anti-amyloid staining revealed β-amyloid deposition within the subiculum of young 5XFAD mice within 48 hrs following infection (Figure 4D). Consistent with HSV1-mediated Aβ seeding, HSV1 immunofluorescence and Aβ signals co-localized. Infected control WT littermates and sham-injected 5XFAD did not show Aβ disposition. Notably, non-lethal HSV1 dosing led to advanced β-amyloid/HSV1 deposition in Layer V of the cortex of 5XFAD mice three-weeks following infection (Figure 4E). β-amyloid deposits initially generated with acute HSV1 infection appear amorphous. However, three weeks post-infection β-amyloid deposits can be observed with immunostaining morphologies that resemble mature senile plaques found in AD brain (Figure 4E).

Our data from cell culture and mouse models are consistent with Aβ-mediated protection from HSV1 and HHV6, involving a classical AMP mechanism of viral entrapment, here, by Aβ fibrils and deposits. These findings indicate that antimicrobial activities of Aβ fibrils and deposits may be important for defending brain against common viral infections. In addition, our findings that viral, as well as bacterial and fungal infections, rapidly and dramatically seed β-amyloid, suggest a broad spectrum of pathogens carry the potential to mediate β-amyloidosis in AD patients.

DISCUSSION

HSV1 is the pathogen most strongly linked to AD with over 100 publications directly or indirectly supporting an association with the disease (with only three that conflict, all published over 12 years ago) (Itzhaki et al., 2016). Most recently, a 16-year long study involving over 33,000 patients found HSV1 infection increases AD risk 2.5 fold (Tzeng et al., 2018). Moreover, long-term administration of anti-herpetic medications appears to reduce AD risk for HSV1 infected patients. Our data reveal a mechanism by which the presence of extracellular herpesviridae viruses may directly seed Aβ deposition as part of AD pathogenesis. Recent findings using 3D human stem cell-derived neural cultures have shown that Aβ generation is sufficient to induce neurofibrillary tangles (Choi et al., 2014). Current data suggest severe neurodegeneration may require the presence of all three major AD pathologies (i.e. plaques, NTFs, and neuroinflammation) (Perez-Nievas et al., 2013). Our current findings are not in and of themselves evidence that herpes infections generate β-amyloidosis in AD brain. However, our findings reveal a plausible mechanism for the genesis of β-amyloidosis driven by the low-grade but persistent viral presence that characterizes chronic symptomatic or subclinical herpesviridae CNS infections. Data showing increased resistance to HSV1 (Figure 1) and accelerated Aβ deposition with infection (Figure 4D-E) in 5XFAD mice is consistent with findings from our cell culture experimental models, in which Aβ antimicrobial activities were mediated by fibrillization. However, for herpesviridae infections in humans, the immediate protection afforded by Aβ entrapment may carry a long-term risk for AD-related pathogenic β-amyloidosis. Possible factors mediating a shift in AD brain from protective to neuropathological Aβ deposition include pathogen virulence and persistence, host genetics, and environmental factors. Importantly, in the antimicrobial protection model, neurodegeneration is not mediated by pathogen activities that directly kill neurons. Rather, Aβ innate immune pathways targeting pathogens mediate the AD neuropathogenesis that leads to widespread neurodegeneration. Thus, our model is consistent with the amyloid cascade hypothesis and overwhelming data showing the primacy of Aβ in AD pathology.

Binding of host brain glycosaminoglycans (GAGs) induces Aβ fibrillization and is most often characterized as an exclusively pathological event associated with β-amyloidosis (van Horssen et al., 2003). However, carbohydrate-binding and fibrillization are considered normally protective activities among AMPs, playing key roles in microbial targeting and agglutination (Chu et al., 2012; Torrent et al., 2012; Tsai et al., 2011,a, b; Yount et al., 2006). Data are consistent with Aβ’s carbohydrate-binding activity mediating targeting and binding of viral surface glycoproteins (Figure 3). Binding of HSV1 and HHV6 induces rapid Aβ fibrillization in cell culture models (Figure 4A). Bound Aβ oligomers and propagating protofibrils on viral surfaces likely sterically inhibit host cell adhesion and herpes glycoprotein activities mediating HSV1 and HHV6 entry (Figure 2). TEM data reveal rapid glycoprotein-induced Aβ fibrillization leads to a network of captured viral particles linked by fibrils within 30 minutes. Finally, in both cell culture and AD mouse models, Aβ fibrillization leads to the sequestering and neutralization of HSV1 and HHV6 viruses in insoluble fibril/virus agglutinates (Figure 4B-E). β-amyloid generated in 3D culture and 5XFAD mouse models during acute HSV1 infection appear amorphous. However, three weeks post-infection deposits in 5XFAD brain show immunohistological morphologies strikingly similar to mature senile plaques in AD brain (Figure 4E). Findings suggest non-lethal HSV1 infection could be useful for accelerating the onset of plaque generation in young 5XFAD, and possibly additional, transgenic AD mouse models.

In our cell culture experimental models, herpes glycoprotein-induced fibrils are generated 1–2 orders of magnitude more rapidly than reported for Aβ fibrillization mediated by GAGs (Castillo et al., 1998). Since viruses minimize extracellular exposure, rapid Aβ fibrillization is likely advantageous for inhibiting and capturing HSV1 and HHV6 before they adhere to host cells. These new data for Aβ-mediated viral agglutination are consistent with findings for bacterial and fungal aggregation (Kumar et al., 2016) and suggest that microbial sugars, rather than host GAGs, may be the normal in vivo target for the heparin-binding activity of Aβ oligomers.

Aβ is an ancient, highly conserved, and widely expressed vertebrate protein. Phylogenetic analyses indicate the herpesviridae family is 300–400 million years old with herpes pathogens identified across all seven classes of vertebrate (McGeoch and Gatherer, 2005). Thus, Aβ and herpes viruses likely have a long history as adversaries. The longevity and remarkable widespread conservation of the human Aβ42 sequence across vertebrates suggest the peptides antiherpetic activities are both efficacious and difficult for herpes viruses to counter. The antimicrobial activities of Aβ are not limited to herpesviridae. Prior in vitro studies have shown that Aβ can also inhibit seasonal and pandemic strains of influenza A virus (White et al., 2014) and a range of bacterial and fungal pathogens (Soscia et al., 2010). Collectively, these findings suggest Aβ oligomerization mediates highly successful innate immune pathways that are intrinsically resistant to evolution of microbial countermeasures. This stands in stark contrast to prevailing views of Aβ oligomerization that characterize self-association and aggregation of the peptide as strictly abnormal and exclusively pathological events. A fuller elucidation of the key role that oligomerization plays in the normal activities of Aβ is likely to help advance our understanding of the etiology and pathogenesis of AD and better inform current and future AD therapeutic strategies aimed at preventing pathological Aβ accumulation.

Finally, with regard to a possible herpes infection-based etiology for AD. The risk for amyloidosis and HSV1 infection in brain both increase with age. The decline of adaptive immunity and blood-brain barrier integrity with advancing senescence is thought to enable HSV1 to travel from the peripheral nervous system to the CNS and be acquired as a new brain infection via the olfactory route. Declining immunity in old age is thought to also allow increase HHV6 replication in brain. However, data supporting a role for herpesviridae, or other infections, is widely held as equivocal. Nonetheless, our identification of a plausible pathway for direct viral mediation of β-amyloid deposition and recent transcriptomic data confirming increased abundance of herpes viruses in AD brain (Readhead et al., 2018) add to mounting evidence that viral and other microbial infections may, indeed, play an important role in the etiology and pathogenesis of this devastating neurodegenerative disease.

EXPERIMENTAL PROCEDURES

AD mouse infection model

Female 5XFAD APP/PS1 transgenic mice (Oakley et al., 2006) that overexpress Aβ42 from inheritable FAD mutant forms of human APP (the Swedish mutation: K670N, M671L; the Florida mutation: I716V; the London mutation: V717I) and PS1 (M146L; L286V) transgenes under transcriptional control of the neuron-specific mouse Thy-1 promoter (Tg6799 line). 5XFAD lines (B6/SJL genetic background) were purchased from Jackson Laboratory and maintained by crossing heterozygous transgenic mice with B6/SJL F1 breeders. All 5XFAD transgenic mice were heterozygotes with respect to the transgene. Animal experiments were conducted in accordance with institutional and NIH guidelines.

Five-to-six-week old mice received bilateral injections (0.20 ml) of HSV1 viron (5 × 106 PFU/μl) suspension at AP, −2.0; ML, +1.6/−1.6; DV, −2.0 using a 5 μl Hamilton syringe with a 30-gauge needle attached to a digital stereotaxic apparatus and an infusion pump at a rate of 0.15 μl/min. After each injection was completed, the needle remained in place for 5 minutes before slow withdrawal. Mice were given food and water on the cage floor starting 24 hrs after the injection. Control sham infections used virus free suspension.

Preparation of synthetic Aβ42

Synthetic Aβ1–42 (Aβ42) and reverse Aβ42–1 (revAβ42) were prepared and purified by Dr. James I. Elliott at Yale University (New Haven, CT) using solid-phase peptide synthesis. Synthetic peptide solutions were generated using established protocols (Kumar et al., 2016). Briefly, bulk powdered peptides were initially incubated in 30% trifluoroethanol to disrupt oligomeric species, lyophilized, and stored (−20 °C) under nitrogen. Prior to experimentation dried peptide films were solubilized in 10 mM NaOH. For experimentation, stock Aβ preparations were serially diluted into culture media. Final Aβ concentration in experimental samples was confirmed by densitometry analysis of anti-Aβ immunoblots using peptide standards of known concentration.

Cell-derived Aβ from conditioned culture media

The stably transformed H4 (H4-Aβ42) (Lewis et al., 2001) and CHO (CHO-CAB) (Hahn et al., 2011) cell lines that express human Aβ have been described previously. H4-Aβ42 cells generate oligomeric Aβ42 while CHO-CAB lines express both Aβ40 and Aβ42 oligomers (Kumar et al., 2016). Naïve H4 (H4-N) and (CHO-N) cell lines were maintained in complete media containing DMEM, 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U penicillin, and 100 μg/ml streptomycin. Complete media for transformed H4-Aβ40 and H4-Aβ42 cells included hygromycin (150 μg/ml) and media for CHO-CAB Zeocin (200 μg/ml) and G418 (200 μg/ml). Confluent host cell cultures were washed twice in PBS, trypsinized, pelleted by centrifugation, washed a second time to remove trypsin and residual antibiotics, and resuspended in antibiotic- free DMEM with 5 % FBS and 2 mM L-glutamine. Cell concentrations were determined by automated cell counter (TC20 BioRad, Hercules CA) and adjusted to 300,000 and 500,000 cells/ml for H4 and CHO lines, respectively. Naïve and transformed culture media were conditioned for 24 hrs with cells before collection. Media samples were used within 12 hrs of collection.

Anti-Aβ immunodepletion

Aβ in conditioned cell culture media was immunodepleted according to established protocols (Kumar et al., 2016). Briefly, protein G Plus Agarose beads coated with mouse monoclonal 4G8 (epitope: Aβ17–24) antibody (Covance, Princeton, NJ) were incubated (8 μg of antibody per ml of media) overnight with samples. Beads were pelleted and soluble fractions, removed, filtered (0.2 μm), and assayed by anti-Aβ ELISA and Western blot to confirm Aβ depletion. (Kumar et al., 2016).

Monolayer cell culture infection model

Experiments used a modified host cell monolayer infection model that has been described previously (Kumar et al., 2016). Trypsinized naïve human neuroglioma (H4-N) host cell were seeded (3 × 105 cells/ml) into 24-well plates and maintained in complete medium containing Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U penicillin, and streptomycin (100 mg/ml) until confluence. For infection experiments, media was replaced with 24 hrs conditioned media from H4-N, H4 Aβ42, or CHO-CAB cella containing HSV1 (3 × 105 PFU). Synthetic A 42 was incubated with HSV1 and host cells in H4-N conditioned media. As a negative control and serial dilution diluent, H4-Aβ42 conditioned media was immunodepleted with anti-Aβ antibodies. Following 2 hours incubation with virus, media was aspirated and cultures washed X3. Fresh media was applied and infected host cells incubated for 20–22 hours. Cells were trypsinized, washed, then fixed in 4% paraformaldehyde for 20 minutes. Cells were then re-suspended in PBS and analyzed for RFP signal on LSRII flow cytometer (BD Biosciences, San Jose, CA)

3D neuronal cell culture system

Procedures for preparation of 3D human stem cell-derived neural cell cultures have been described previously (Choi et al., 2014). Briefly, ReNcell VM human neural precursor cells (Billerica, MA, USA) were added to BD Matrigel (BD Biosciences, San Jose, CA) containing ReN cell differentiation medium and transferred to the wells of 96-well culture plates (100 μl per well containing 2 ×105 cells). The 3D-plated cells were differentiated for 3 weeks prior to experimentation.

For pathogen exposure, 100 μl of media was removed and replace with equal volume media containing either HSV1 or HHV6 (3 ×104 PFU). Cells were exposed to viral pathogens for 48 to 72 hrs and then washed and fixed overnight in 4% paraformaldehyde for analysis.

Aβ-binding ELISA.

The wells of 96-well plates were incubated (80°C) with live virus (3×105 PFU/well) in distilled water (50 μl/well) until wells were dry (1–2 hrs). Wells were then washed X5 with Tris buffered saline (TBS) containing 0.05% triton X-100 (TBST) to remove unattached virus and then blocked with 5 % BSA in TBS for 2 hrs at RT. Samples for testing were incubated (1hr at 37° C) in virus-coated wells (100 μl /well) and following washing with TBST, bound Aβ detected immunochemically by incubation (overnight at 4° C) with α-Aβ42-HRP (Covance, Princeton, NJ) and development with chemiluminescence reagent (Pierce, Rockford IL). For glycoprotein binding experiments, virus-coated wells were pre-incubated (4°C overnight) with antibodies (1:1000) against glycoproteins B (α-gB), C (α-gC), D (α-gD), E (α-gE), G (α-gG), or H (α-gH)

Tissue preparation and sectioning.

For immunofluorescence, mice were deeply anesthetized with isoflurane, and perfused transcardially with 4% paraformaldehyde in cold PBS when possible. Brains were post-fixed overnight and then transferred into a 30% sucrose solution until sedimented. Sagittal sections (30 μm) were cut from an ice-cooled block using a sliding microtome (Leica, Wetzlar, Germany). Sections were stored at −20°C in cryoprotective buffer containing 28% ethylene glycol, 23% glycol and 0.05 M phosphate until processing for analysis.

Immunofluorescence labeling

Immunofluorescence labeling was performed as described previously (Oakley et al., 2006). Briefly, sections or wells were probed with anti-Aβ42 monoclonal rabbit (ThermoFisher Scientific, Boston, MA) followed by anti-rabbit Alexafluor 405 secondary (Invitrogen, 8889S) antibodies. For β-amyloid detection, free-floating sections or wells were incubated (8 min) with 0.002% Thioflavin S in TBS, rinsed twice for 1 min in 50% ethanol, washed for 5 min in TBS, and mounted with Prolong Gold antifade reagent (Life Technologies) or the wells were maintained in TBS. Stained sections were analyzed by fluorescence confocal microscopy on a Nikon C2s laser scanning microscope (Nikon, Tokyo, Japan).

Viral agglutination and TEM analysis.

HSV1 or HHV6 incubated in conditioned culture media for 15, 30, or 60 minutes at RT. Viral aggregates were pelleted, resuspended in PBS (5 μl), and absorbed to Formvar carbon coated copper grids (FCF100-Cu, Electron Microscopy Sciences, PA). Grids were blocked with 1 % BSA in PBS (kept covered for 10 min at RT) then incubated (30 min at RT) with mAb 4G8 (1:1000) in blocking buffer. Grids were washed with PBS and incubated with goat anti-mouse-IgG antibody covalently linked to nanogold particles. Following three 5 min PBS washes and four 10 min water washes, specimens were fixed with 1 % glutaraldehyde (10 min at RT). Specimens were washed with water, stained with uranyl acetate, and then viewed using a JEM-1011 Transmission Electron Microscope (JEOL Institute, Peabody, MA).

Statistical analysis.

Statistical analyses were performed with Prism software (version 7.0) using two tailed t-tests to compare arithmetic means. Survival curves were compared using Log-rank (Mantel-Cox) test and confirmed by Gehan-Breslow-Wilcoxon test. P values < 0.05 were considered statistically significant.

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rudolph Tanzi (tanzi@helix.mgh.harvard.edu)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Female 5XFAD mice (B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax, 34840-JAX), 5–6 weeks old, and WT littermates (B6SJLF1/J, 100012) were used for in vivo experiments. Mice were group-housed and kept on a 12 hr light/dark cycle with ad libitum access to food and water. All procedures in this study were conducted in accordance with approvals granted by Institutional Animal Care and Use Committee at MGH and NIH guidelines.

Ren-VM (male), VERO (female), H4-Naïve or Aβ42 (male), and CHO-Naïve or CAB (female) cells were all housed in 37°C incubators with 5% CO2 and ≥ 93% humidity. H4-Aβ42 and CHO-CAB were maintained by selection antibiotics and all cell lines were authenticated by morphology checks at both cell seeding and confluency growth stages.

METHOD DETAILS

AD mouse infection model

Female 5XFAD APP/PS1 transgenic mice (Oakley et al., 2006) that overexpress Aβ42 from inheritable FAD mutant forms of human APP (the Swedish mutation: K670N, M671L; the Florida mutation: I716V; the London mutation: V717I) and PS1 (M146L; L286V) transgenes under transcriptional control of the neuron-specific mouse Thy-1 promoter (Tg6799 line). 5XFAD lines (B6/SJL genetic background) were purchased from Jackson Laboratory and maintained by crossing heterozygous transgenic mice with B6/SJL F1 breeders. All 5XFAD transgenic mice were heterozygotes with respect to the transgene.

Five-to-six-week old mice received bilateral injections (0.20 ml) of HSV1 viron (5 × 106 PFU/μl) suspension at AP, −2.0; ML, +1.6/−1.6; DV, −2.0 using a 5 μl Hamilton syringe with a 30-gauge needle attached to a digital stereotaxic apparatus and an infusion pump at a rate of 0.15 μl/min. After each injection was completed, the needle remained in place for 5 minutes before slow withdrawal. Mice were given food and water on the cage floor starting 24 hrs after the injection. Control sham infections used virus free suspension.

Preparation of synthetic Aβ42

Synthetic Aβ1–42 (AE42) and reverse Aβ42–1 (revAE42) were prepared and purified by Dr. James I. Elliott at Yale University (New Haven, CT) using solid-phase peptide synthesis. Synthetic peptide solutions were generated using estAβlished protocols (Kumar et al., 2016). Briefly, bulk powdered peptides were initially incubated in 30% trifluoroethanol to disrupt oligomeric species, lyophilized, and stored (−20 °C) under nitrogen. Prior to experimentation dried peptide films were solubilized in 10 mM NaOH. For experimentation, stock AE preparations were serially diluted into culture media. Final AE concentration in experimental samples was confirmed by densitometry analysis of anti-AE immunoblots using peptide standards of known concentration.

Cell-derived Aβ from conditioned culture media

The stably transformed H4 (H4-Aβ42) (Lewis et al., 2001) and CHO (CHO-CAB) (Hahn et al., 2011) cell lines that express human Aβ have been described previously. H4-Aβ42 cells generate oligomeric Aβ42 while CHO-CAB lines express both Aβ40 and Aβ42 oligomers (Kumar et al., 2016). Naïve H4 (H4-N) and (CHO-N) cell lines were maintained in complete media containing DMEM, 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U penicillin, and 100 μg/ml streptomycin. Complete media for transformed H4-Aβ40 and H4-Aβ42 cells included hygromycin (150 μg/ml) and media for CHO-CAB Zeocin (200 μg/ml) and G418 (200 μg/ml). Confluent host cell cultures were washed twice in PBS, trypsinized, pelleted by centrifugation, washed a second time to remove trypsin and residual antibiotics, and resuspended in antibiotic- free DMEM with 5 % FBS and 2 mM L-glutamine. Cell concentrations were determined by automated cell counter (TC20 BioRad, Hercules CA) and adjusted to 300,000 and 500,000 cells/ml for H4 and CHO lines, respectively. Naïve and transformed culture media were conditioned for 24 hrs with cells before collection. Media samples were used within 12 hrs of collection.

Anti-Aβ immunodepletion

Aβ in conditioned cell culture media was immunodepleted according to estAβlished protocols (Kumar et al., 2016). Briefly, protein G Plus Agarose beads coated with mouse monoclonal 4G8 (epitope: Aβ17–24) antibody (Covance, Princeton, NJ) were incubated (8 μg of antibody per ml of media) overnight with samples. Beads were pelleted and soluble fractions, removed, filtered (0.2 μm), and assayed by anti-Aβ ELISA and Western blot to confirm Aβ depletion. (Kumar et al., 2016).

Monolayer cell culture infection model

Experiments used a modified host cell monolayer infection model that has been described previously (Kumar et al., 2016). Trypsinized naïve human neuroglioma (H4-N) host cell were seeded (3 × 10 cells/ml) into 24-well plates and maintained in complete medium containing Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U penicillin, and streptomycin (100 mg/ml) until confluence. For infection experiments, media was replaced with 24 hrs conditioned media from H4-N, H4-Aβ42, or CHO-CAB cells containing HSV1 (3 × 105 PFU). Synthetic Aβ42 was incubated with HSV1 and host cells in H4-N conditioned media. As a negative control and serial dilution diluent, H4-Aβ42 conditioned media was immunodepleted with anti-Aβ antibodies. Following 2 hours incubation with virus, media was aspirated and cultures washed X3. Fresh media was applied and infected host cells incubated for 20–22 hours. Cells were trypsinized, washed, then fixed in 4% paraformaldehyde for 20 minutes. Cells were then re-suspended in PBS and analyzed for RFP signal on LSRII flow cytometer (BD Biosciences, San Jose, CA)

3D neuronal cell culture system

Procedures for preparation of 3D human stem cell-derived neural cell cultures have been described previously (Choi et al., 2014). Briefly, ReNcell VM human neural precursor cells (Billerica, MA, USA) were added to BD Matrigel (BD Biosciences, San Jose, CA) containing ReN cell differentiation medium and transferred to the wells of 96-well culture plates (100 μl per well containing 2 ×105 cells). The 3D-plated cells were differentiated for 3 weeks prior to experimentation.

For pathogen exposure, 100 μl of media was removed and replace with equal volume media containing either HSV1 or HHV6 (3 ×104 PFU). Cells were exposed to viral pathogens for 48 to 72 hrs and then washed and fixed overnight in 4% paraformaldehyde for analysis.

Aβ-binding ELISA

The wells of 96-well plates were incubated (80°C) with live virus (3×105 PFU/well) in deionized water (50 μl/well) until wells were dry (1–2 hrs). Wells were then washed X5 with Tris buffered saline (TBS) containing 0.05% triton X-100 (TBST) to remove unattached virus and then blocked with 5 % BSA in TBS for 2 hrs at RT. Samples for testing were incubated (1hr at 37° C) in virus-coated wells (100 μl /well) and following washing with TBST, bound Aβ detected immunochemically by incubation (overnight at 4° C) with α-Aβ42-HRP (Covance, Princeton, NJ) and development with chemiluminescence reagent (Pierce, Rockford IL). For glycoprotein binding experiments, virus-coated wells were pre-incubated (4°C overnight) with antibodies (1:1000) against glycoproteins B (α-gB), C (α-gC), D (α-gD), E (α-gE), G (α-gG), or H (α-gH)

Tissue preparation and sectioning

For immunofluorescence, mice were deeply anesthetized with isoflurane, and perfused transcardially with 4% paraformaldehyde in cold PBS when possible. Brains were post-fixed overnight and then transferred into a 30% sucrose solution until sedimented. Sagittal sections (30 μm) were cut from an ice-cooled block using a sliding microtome (Leica, Wetzlar, Germany). Sections were stored at −20°C in cryoprotective buffer containing 28% ethylene glycol, 23% glycol and 0.05 M phosphate until processing for analysis.

Immunofluorescence labeling

Immunofluorescence labeling was performed as described previously (Oakley et al., 2006). Briefly, sections or wells were probed with anti-Aβ42 monoclonal rabbit (ThermoFisher Scientific, Boston, MA) followed by anti-rabbit Dylight 405 secondary (Jackson ImmunoResearch, 711-475-152) antibodies. For β-amyloid detection, free-floating sections or wells were incubated (8 min) with 0.002% Thioflavin S in TBS, rinsed twice for 1 min in 50% ethanol, washed for 5 min in TBS, and mounted with Prolong Gold antifade reagent (Life Technologies) or the wells were maintained in TBS. Stained sections were analyzed by fluorescence confocal microscopy on a Nikon C2s laser scanning microscope (Nikon, Tokyo, Japan).

Viral agglutination and TEM analysis

HSV1 or HHV6 incubated in conditioned culture media for 15, 30, or 60 minutes at RT. Viral aggregates were pelleted, resuspended in PBS (5 μl), and absorbed to Formvar carbon coated copper grids (FCF100-Cu, Electron Microscopy Sciences, Hatfield, PA). Grids were blocked with 1 % BSA in PBS (kept covered for 10 min at RT) then incubated (30 min at RT) with mAb 4G8 (1:1000) in blocking buffer. Grids were washed with PBS and incubated with goat anti-mouse-IgG antibody covalently linked to nanogold particles. Following three 5 min PBS washes and four 10 min water washes, specimens were fixed with 1 % glutaraldehyde (10 min at RT). Specimens were washed with water, stained with uranyl acetate, and then viewed using a JEM-1011 Transmission Electron Microscope (JEOL Institute, Peabody, MA).

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were performed with Prism software (version 7.0) using two tailed t-tests to compare arithmetic means. Survival curves were compared using Log-rank (Mantel-Cox) test and confirmed by Gehan-Breslow-Wilcoxon test. P values < 0.05 were considered statistically significant.

Supplementary Material

SF1-3

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Anti-B-Amyloid, 17–24 Antibody 4G8 BioLegend RRID:AB_2564633
HRP anti-B-Amyloid, 1–16 Antibody BioLegend RRID:AB_2564667
Anti-AB42 Monoclonal Rabbit Antibody ThermoFisher Scientific Cat. No. 700254
DyLight 405 Donkey Anti-Rabbit IgG Jackson ImmunoResearch Labs RRID:AB_2340616
HSV Type 1/2 gB Monoclonal Antibody (T111) ThermoFisher Scientific RRID:AB_2214680
HSV Type 1 gC Monoclonal Antibody (T96) ThermoFisher Scientific RRID:AB_1075604
HSV Type 1/2 gD Monoclonal Antibody (G610D) ThermoFisher Scientific RRID:AB_1017563
Anti-HSV1 gE Envelope Protein Antibody [9H3] abcam RRID:AB_305533
Anti-HSV1 gG Envelope Protein Antibody [M612451] abcam RRID:AB_880671
Anti-HSV1 gH Antibody [BBH1] abcam RRID:AB_10866046
Bacterial and Virus Strains
HSV1 Breakefield Lab N/A
HHV6A Public Health England Cat. No. 0003121v
HHV6B Public Health England Cat. No. 0006111v
Chemicals, Peptides, and Recombinant Proteins
Synthetic AB42 Elliott Lab N/A
Experimental Models: Cell Lines
CHO-N ATCC RRID:CVCL_C462
CHO-CAB Moir Lab N/A
H4-N ATCC RRID:CVCL_1239
H4–42 Moir Lab N/A
VERO ATCC RRID:CVCL_0059
ReN VM Millipore Sigma RRID:CVCL_E921
Experimental Models: Organisms/Strains
5XFAD Mice The Jackson Laboratory RRID:MMRRC_0348 40-JAX
WT Mice The Jackson Laboratory RRID:IMSR_JAX:100 012
Other
Formvar carbon coated copper grids Electron Microscopy Sciences Cat. No. FCF100Cu
Pierce Protein G Plus Agarose Beads ThermoFisher Scientific Cat. No. 22851
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Highlights.

  • Human Aβ protects against herpesviridae in AD mouse and 3D human neuronal cell cultures

  • Fibrilization mediates Aβ antiherpetic activities, entrapping viruses in β-amyloid

  • Herpesviridae infections dramatically accelerate Aβ-amyloidosis in AD models

ACKNOWLEDGMENTS:

The authors would like to thank Todd Golde (University Florida, Gainesville, USA) for the Aβ-expressing transfected cell line and Prof Cornel Fraefel (Virologisches Institut, Zurich, Switzerland) for RFP labeled HSV1 virus. No patents have been filed in associated with this work. The authors have also not been engaged for consulting purposes nor own companies related to this work.

Footnotes

Data Availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files.

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Supplementary Materials

SF1-3
NIHMS982565-supplement-SF1-3.pdf (6.5MB, pdf)

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