Alzheimer's disease is a progressive neurodegenerative disease characterized neuropathologically by presence of extracellular amyloid plaques composed of fibrillar amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles. Post-mortem and in vivo studies implicate HSV-1 infection in the brain as a precipitating factor in disease/pathology initiation. HSV-1 infection of two-dimensional (2D) neuronal cultures causes intracellular accumulation of Aβ42 peptide, but these 2D models do not recapitulate the three-dimensional (3D) architecture of brain tissue. We employed human induced pluripotent stem cells (hiPSCs) to compare patterns of Aβ42 accumulation in HSV-1 infected 2D (neuronal monolayers) and 3D neuronal cultures (brain organoids). Akin to prior studies, HSV-1-infected 2D cultures showed Aβ42 immunoreactivity in cells expressing the HSV-1 antigen ICP4 (ICP4+). Conversely, accumulation of Aβ42 in ICP4+ cells in infected organoids was rarely observed. These results highlight the importance of considering 3D cultures to model host-pathogen interaction. IMPORTANCE The “pathogen” hypothesis of Alzheimer’s disease (AD) proposes that brain HSV-1 infection could be an initial source of amyloid beta (Aβ) peptide-containing amyloid plaque development. Aβ accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures, as well as in HSV-1-infected 3D neuronal culture models. The current study extends these findings by showing different patterns of Aβ42 accumulation following HSV-1 infection of 2D compared to 3D neuronal cultures (brain organoids). Specifically, 2D neuronal cultures showed Aβ42-immunoreactivity mainly in HSV-1-infected cells and only rarely in uninfected cells or infected cells exposed to antivirals. Conversely, 3D brain organoids showed accumulation of Aβ42 mainly in non-infected cells surrounding HSV-1-infected cells. We suggest that because brain organoids better recapitulate architectural features of a developing brain than 2D cultures, they may be a more suitable model to investigate the involvement of HSV-1 in the onset of AD pathology.

Background Drug repurposing is a cost-effective strategy to identify drugs with novel effects. We searched for drugs exhibiting inhibitory activity to Herpes Simplex virus 1 (HSV-1). Our strategy utilized gene expression data generated from HSV-1-infected cell cultures which was paired with drug effects on gene expression. Gene expression data from HSV-1 infected and uninfected neurons were analyzed using BaseSpace Correlation Engine (Illumina®). Based on the general Signature Reversing Principle (SRP), we hypothesized that the effects of candidate antiviral drugs on gene expression would be diametrically opposite (negatively correlated) to those effects induced by HSV-1 infection. Results We initially identified compounds capable of inducing changes in gene expression opposite to those which were consequent to HSV-1 infection. The most promising negatively correlated drugs (Valproic acid, Vorinostat) did not significantly inhibit HSV-1 infection further in African green monkey kidney epithelial cells (Vero cells). Next, we tested Sulforaphane and Menadione which showed effects similar to those caused by viral infections (positively correlated). Intriguingly, Sulforaphane caused a modest but significant inhibition of HSV-1 infection in Vero cells (IC50 = 180.4 µM, p = 0.008), but exhibited toxicity when further explored in human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells. Conclusions These results reveal the limits of the commonly used SRP strategy when applied to the identification of novel antiviral drugs and highlight the necessity to refine the SRP strategy to increase its utility.

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