Modeling Aβ42 Accumulation in Response to Herpes Simplex Virus 1 Infection: 2D or 3D?

Abstract

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.

FIG 1

Immunofluorescence for amyloid-β42 and the HSV-1 antigen ICP4 in uninfected and HSV-1-infected hiPSC-derived neurons. hiPSC-derived 2D neuronal cultures were infected at MOIs of 1 to 0.3. Neurons were also infected at an MOI of 0.3 in the presence of antivirals 5BVdU and IFN-α. (a to f) Microphotographs depicting immunostaining of Aβ42 and the HSV-1 antigen ICP4 in uninfected neurons (a), neurons infected at an MOI of 0.3 in the presence of 5BVdU and IFN-α (b), and neurons acutely infected at MOIs of 1 to 0.3 (c to f). Scale bar is 25 μm (a and c, right), 10 μm (b; c, left; d; e; and f). Quantification of Aβ42 immunofluorescence intensity using ImageJ (g). The increase in Aβ42 fluorescence intensity in infected neurons compared to uninfected neurons was assessed using Student's t test. Error bars represent standard deviations.

FIG 2

Characterization of 3D brain organoids. (a) Hematoxylin and eosin staining of 9-week-old organoids. (b to j) Immunostaining of 8-μm-thick sections of organoids with antibodies recognizing FOXG1 (b), SOX2 (c), vimentin (d), TBR2 (e), Tuj/CUX2 (f), TBR1 (g), calbindin (h), CTIP2 (i), and CTIP2/SATB2 (j). Nuclei were counterstained with Hoechst 33342. Scale bars are 100 μm (a), 75 μm (b, d, and j), 50 μm (c and e), and 250 μm (f, g, h, and i).

FIG 3

Immunofluorescence for amyloid-β42 and the HSV-1 antigen ICP4 in uninfected and HSV-1-infected 3D brain organoids. Twelve-week-old organoids were infected singularly with 3,000 PFU in the presence or absence of antivirals 5BVdU and IFN-α. After 2 h, the inocula were removed and the organoids were cultured in the presence or absence of antivirals. After 72 h, the organoids were processed for immunofluorescence. (a) Microphotographs of 8-μm-thick sections of HSV-1-infected (a) and uninfected (b) organoids immunostained with ICP4/MAP2 and infected organoids in the presence (c) or absence (d to l) of antivirals with ICP4/Aβ42. Nuclei were counterstained with Hoechst 33342. ICP4⁺ cells exhibiting intracellular Aβ42 immunoreactivity are indicated by arrows. The insets contain enlarged details. Scale bars are 250 μm (a), 25 μm (b, c, d, e, g, and l), and 10 μm (f, i, j, and k).