Volume 99, Issue 1, Pages 56-63.e3 (July 2018) Alzheimer’s Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection  William A. Eimer, Deepak Kumar Vijaya Kumar, Nanda Kumar Navalpur Shanmugam, Alex S. Rodriguez, Teryn Mitchell, Kevin J. Washicosky, Bence György, Xandra O. Breakefield, Rudolph E. Tanzi, Robert D. Moir  Neuron  Volume 99, Issue 1, Pages 56-63.e3 (July 2018) DOI: 10.1016/j.neuron.2018.06.030 Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 Aβ42 Expression Increases Host Survival in an HSV1 Encephalitis 5XFAD Mouse Model Female transgenic mice (5XFAD), 5–6 weeks old, 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. Neuron 2018 99, 56-63.e3DOI: (10.1016/j.neuron.2018.06.030) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Cell-Derived Aβ42 Oligomers Mediate HSV1 Resistance in Cell Culture Monolayer Infection Models Confluent human H4-N cell monolayers were co-incubated (2 hr) 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 naive 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 naive and transformed signal were by t test. Data are representative of at least six independent experiments for each condition. Neuron 2018 99, 56-63.e3DOI: (10.1016/j.neuron.2018.06.030) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 Aβ Heparin-Binding Activity Mediates Binding of Herpesviridae Glycoproteins 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), and then incubated with H4-Aβ42 conditioned media. Signal shown as percentage of naive 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 eight independent experiments. Neuron 2018 99, 56-63.e3DOI: (10.1016/j.neuron.2018.06.030) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Aβ Fibrilization Mediates Viral Entrapment in 3D Human Stem Cell-Derived Neural Cell Culture and 5XFAD Mouse Infection Models Aβ fibrilization 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 hr) with virus and fluorescence signals captured under (B) low or (C) high magnification. (D and E) Wild-type (WT) and transgenic AD (5XFAD) female mice, 5–6 weeks old, 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 hr 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. Neuron 2018 99, 56-63.e3DOI: (10.1016/j.neuron.2018.06.030) Copyright © 2018 Elsevier Inc. Terms and Conditions