SARS-CoV-2 infects neurons and induces neuroinflammation in a non-human primate model of COVID-19

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), can induce a plethora of neurological complications in some patients. However, it is still under debate whether SARS-CoV-2 directly infects the brain or whether CNS sequelae result from systemic inflammatory responses triggered in the periphery. By using high-resolution microscopy, we investigated whether SARS-CoV-2 reaches the brain and how viral neurotropism can be modulated by aging in a non-human primate model of COVID-19. Seven days after infection, SARS-CoV-2 was detected in the olfactory cortex and interconnected regions and was accompanied by robust neuroinflammation and neuronal damage exacerbated in aged, diabetic animals. Our study provides an initial framework for identifying the molecular and cellular mechanisms underlying SARS-CoV-2 neurological complications, which will be essential to reducing both the short- and long-term burden of COVID-19.

Keywords: CP: Microbiology; CP: Neuroscience; NHP; astrocytes; coronavirus; macaque; microglia; neurotropism; rhesus.

Graphical abstract

Figure 1

SARS-CoV2 preferentially infects neurons and spreads faster throughout the olfactory cortex of aged, infected monkeys (A and B) Quantitative quadruple staining combining markers for neuron (NeuN), microglia (Iba1), and astrocytes (GFAP) with SARS-CoV2 nucleocapsid protein (N ptn) was performed in the primary olfactory cortex of young and aged infected monkeys. (C) Internalized N ptn volume was calculated in 3D and divided by the total 3D volume obtained for each infected cell type analyzed. (D) The neurotropic potential of SARS-CoV2 was investigated in the primary olfactory cortex (blue; piriform cortex [PC], olfactory tubercle [OT], and entorhinal cortex [EC]) and the prefrontal secondary olfactory region, the orbitofrontal cortex (OFC; green). (E) Representative micrographs and 3D volume reconstruction show spike (Spk) protein (purple) and dsRNA (red) expression across several olfactory regions. (F and G) Quantification of the intraneuronal levels of dsRNA (F) and Spk protein (G) demonstrates that SARS-CoV-2 spreads faster in aged animals compared with young, infected controls. Scale bar, 50 μm. ^∗∗p < 0.01, ^∗∗∗p < 0.001, two-way ANOVA, Sidak’s post hoc test. Numerical data are represented as mean ± SEM. See also Figures S2 and S3 and Video S1.

Figure 2

The highly connected olfactory cortex shows a robust inflammatory response following SARS-CoV-2 infection (A and B) Unbiased 3D microscopic quantification shows a significant increase in astrocyte recruitment (GFAP+) following SARS-CoV-2 infection. (C and E) In addition, a robust increase in general (Iba1) and activated (HLA-DR) microglia markers are also associated with aging. (F) HLA-DR+ reactive microglia were also found associated with degraded myelin basic protein (dgMBP), resulting in reduced normal myelin basic protein (MBP) expression in aged-infected animals in comparison with aged controls. (G) To further analyze neuron-microglia dynamics across the experimental groups, we combined general markers for neurons (pan-NF) and microglia (Iba1) with the post-synaptic marker PSD95 in the primary olfactory region. (H–L) Analysis of PSD95 puncta density (H), microglial total cell volume (I), pan-NF microglial engulfment (J), microglia soma volume (K), and PSD95 microglial engulfment (L) demonstrates a robust phagocytic response observed in the brain following SARS-CoV-2 infection, especially in the older infected population. Scale bars, 200 μm (A–F), 10 μm, and 5 μm (3D) (G). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way ANOVA, Tukey’s post hoc test. Numerical data are represented as mean ± SEM. See also Figures S4–S7 and Video S1.

Figure 3

SARS-CoV-2 infection induces blood vessel disruption, reactive neutrophil recruitment, and ACE2 receptor downregulation (A–D) Airyscan super-resolution microscopy was applied to quantify ACE2 and Spk protein expression and binding within olfactory neurons (pan-NF maker) across the different experimental groups. (E) ACE2 expression is substantially reduced in the neurons of aged, infected animals compared with young, infected controls. (F) In addition, around 15%–40% of the remaining receptors colocalize with Spk protein in both infected groups. Multiple structural abnormalities in the BBB were observed across the olfactory cortex of infected monkeys. (G) Neurons (NeuN) and astrocytes (GFAP) present high levels of ACE2 expression and are also the major cell types expressing Spk protein. (H) Notably, disrupted blood vessels are associated with activated neutrophils (MPO) and robust recruitment of reactive astrocytes. (I) Altered BBB was also confirmed using aquaporin 4 (AQ4), a water channel expressed in astrocytic endfeet and involved in edema formation. (J and K) Additionally, the identity of neutrophils was confirmed using the CD66a marker, recruited to the injury. (L) Isolated blood vessels from surrounding meninges also show viral expression. Scale bars, 10 μm (A–D), 50 μm (G–K), and 5 μm (L). ^∗p < 0.05, ^∗∗p < 0.01, one-way ANOVA, Tukey’s post hoc test. Numerical data are represented as mean ± SEM. See also Figure S6 and Video S1.

Figure 4

Summary of the anatomical localization, cell type, and viral protein spread observed in SARS-CoV-2-infected monkeys Schematic representation of the components of the olfactory circuit theorized to serve as the SARS-CoV-2-spreading pathway during the acute stage of the disease. The major viral proteins analyzed in this study were contained within the neural circuitry of the primary olfactory cortex, extending to the medial part of the OFC, a secondary olfactory region, only in aged monkeys. A robust immune response characterized by systemic neutrophil recruitment associated with disrupted blood vessels and local microglia and astrocyte activation was observed across all regions analyzed, including part of the hippocampal formation. I, cortical layer I; II, cortical layer II; III, cortical layer III; IV, cortical layer IV; V, cortical layer V; VI, cortical layer VI; ACE2, angiotensin-converting enzyme 2; dsRNA, double-stranded RNA; HF-CA1, hippocampal formation, CA1 field; mOFC, orbitofrontal cortex, medial part; OT, olfactory tubercle; PC, piriform cortex; rEC, entorhinal cortex, rostral subdivision; Sub, subicular complex.