Neuroinvasion of SARS-CoV-2 in human and mouse brain

Eric Song, Ce Zhang, Benjamin Israelow, Alice Lu-Culligan, Alba Vieites Prado, Sophie Skriabine, Peiwen Lu, Orr-El Weizman, Feimei Liu, Yile Dai, Klara Szigeti-Buck, Yuki Yasumoto, Guilin Wang, Christopher Castaldi, Jaime Heltke, Evelyn Ng, John Wheeler, Mia Madel Alfajaro, Etienne Levavasseur, Benjamin Fontes, Neal G Ravindra, David Van Dijk, Shrikant Mane, Murat Gunel, Aaron Ring, Syed A Jaffar Kazmi, Kai Zhang, Craig B Wilen, Tamas L Horvath, Isabelle Plu, Stephane Haik, Jean-Leon Thomas, Angeliki Louvi, Shelli F Farhadian, Anita Huttner, Danielle Seilhean, Nicolas Renier, Kaya Bilguvar, Akiko Iwasaki, Eric Song, Ce Zhang, Benjamin Israelow, Alice Lu-Culligan, Alba Vieites Prado, Sophie Skriabine, Peiwen Lu, Orr-El Weizman, Feimei Liu, Yile Dai, Klara Szigeti-Buck, Yuki Yasumoto, Guilin Wang, Christopher Castaldi, Jaime Heltke, Evelyn Ng, John Wheeler, Mia Madel Alfajaro, Etienne Levavasseur, Benjamin Fontes, Neal G Ravindra, David Van Dijk, Shrikant Mane, Murat Gunel, Aaron Ring, Syed A Jaffar Kazmi, Kai Zhang, Craig B Wilen, Tamas L Horvath, Isabelle Plu, Stephane Haik, Jean-Leon Thomas, Angeliki Louvi, Shelli F Farhadian, Anita Huttner, Danielle Seilhean, Nicolas Renier, Kaya Bilguvar, Akiko Iwasaki

Abstract

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Conflict of interest statement

Declaration of Interests

None of the authors declare interests related to the manuscript.

Figures

Figure 1:. SARS-CoV-2 infects human brain organoids…
Figure 1:. SARS-CoV-2 infects human brain organoids and induces cell death.
Human brain organoids were infected with SARS-CoV-2 and collected at 24 hours post infection (hpi) or 96 hpi to analyze for different cellular markers. (A) Images of brain organoids looking at SARS-CoV-2 infection (in red) 24 hpi (See also Figure S2C for additional images). (B) Images of brain organoids looking at SARS-CoV-2 infection (in red) 96 hpi (See also Figure S2D for additional images). (C) Quantification of SARS-CoV-2 positive cells in a cortical region of organoids (A-B). (D) Tiled image of 96 hpi organoid. (E) Electron microscopy image of SARS-CoV-2 viral particles in brain organoids (See also Figure S3 for uncropped and additional images). (F) Organoids were stained with TUNEL to evaluate cell death at 96hpi. (G) Quantification of SARS-CoV-2 and TUNEL double positive (yellow) or SARS-CoV-2 negative, TUNEL positive (green) cells over total TUNEL positive cells. (H) Quantification of SARS-CoV-2 and TUNEL double positive (yellow) or SARS-CoV-2 positive, TUNEL negative (red) cells over total SARS-CoV-2 positive cells. (I) Correlation between the frequency of TUNEL positive cells and presence SARS-CoV-2 in different regions of the organoid. (I) Representative image of TUNEL and SARS-CoV-2 staining showing high-density SARS-CoV-2 region (yellow box) and low-density SARS-CoV-2 region (white box) in the same plane. All experiments were performed with unique organoid n of 4 per condition, from the same culturing batch, with images from n = 12 cortical regions with two IPSC lines, and student’s t-test was performed (****, P<0.0001).
Figure 2:. Neuronal cells undergo a unique…
Figure 2:. Neuronal cells undergo a unique metabolic response to SARS-CoV-2 infections.
Brain organoids were infected with SARS-CoV-2 and sequenced with 10x single cell sequencing strategies. (A) Projection of cells with SARS-CoV-2 transcripts (in red) onto the UMAP. Percentage of infected cells in each cluster. (B) Change in population representation of example clusters after infection (refer See also Figure S5 for all clusters). (C) Validation of neuronal subtypes using CTIP2, PAX6 and TBR1 antibodies for confocal imaging. (D) Differentially expressed genes (DEGs) from brain organoids infected with ZIKV (Watnabe et al 2017 Cell Reports) were compared with DEGs from SARS-CoV-2 infected organoids. (E) Enriched gene ontology terms (geneontology.com) for upregulated genes from (D). (F) Enriched GO terms in SARS-CoV-2 infected cells (top) and SARS-CoV-2 negative bystander cells from 96 hpi organoids (bottom). (G) Heatmap of genes from metabolic pathways. (H) HIF1A staining of brain organoids that were mock infected versus 96hpi. (I) Quantification of HIF1A positive cells in SARS-CoV-2 infected organoids. Single cell RNA-seq was performed in duplicates with one IPSC line (Y6). HIF1A staining was performed with unique organoid n of 4 per condition, from the same culturing batch, with images from n = 12 cortical regions with two IPSC lines, and student’s t-test was performed (****, P<0.0001).
Figure 3:. SARS-CoV-2 neural infection depends on…
Figure 3:. SARS-CoV-2 neural infection depends on ACE2 and can be neutralized by anti-spike antibodies found in CSF of COVID-19 patients.
(A) Immunofluorescence staining of ACE2 in brain organoids. (B) Immunofluroescence staining of organoids pre-incubated with isotype antibodies (top row) or anti-ACE2 antibodies (bottom row) and infected with SARS-CoV-2. (C) Schematic showing collection of clinical lumbar puncture from patients with and without COVID-19 for assays shown in (D-F). (E) Immunofluroescence staining of organoids infected with SARS-CoV-2 preincubated with CSF from health patients (top row) or CSF from COVID-19 patients (bottom row). (F) Quantification of figures from (C and E). All experiments were performed with unique organoid n of 4 per condition, from the same culturing batch, with images from n = 12 cortical regions with two IPSC lines, and student’s t-test was performed (****, P<0.0001).
Fig. 4:. SARS-CoV-2 replicates efficiently in the…
Fig. 4:. SARS-CoV-2 replicates efficiently in the brain of mice and can cause central nervous system specific lethality.
(A-C) Mice expressing human ACE2 under the K18 promoter (K18-hACE2) were infected with SARS-CoV-2 intranasally, and brains of the mice were collected at days 2, 4 and 7 post infection for (A) qPCR or (B) plaque assay. (C-E) iDISCO+ whole brain immunolabeling against the nucleocapsid protein of SARS-CoV2 7 days after an intranasal infection, shown as 300μm projection planes. (C) Dorsal, ventral and sagittal projections showing widespread distribution of the virus in the forebrain with patches of high viral density in the cortex (arrow). The virus is not detected in the cerebellum, except for the pial meninges and DCNs. (D) 300μm deep projection planes in the cortex showing cortical patches of viral expression (arrowhead), a reduced infection of the cells in the layer 4, and expression in pyramidal neurons (arrow). (E) ClearMap analysis of the infected cells distribution (n=3), registered to the Allen Brain Atlas, showing the wide distribution of the virus across brain regions, with a few regions with lower densities, among which the DG, GPi, CA3, cortical layer 4 and VMH. (F-G) Analysis of the vascular network using ClearMap and iDISCO+ 7 days after intranasal infection by mapping of the vascular network with a co-labeling of the N protein. Planes at the level of the Nose somatosensory cortex are shown. (F) Control uninfected brains. Branch point densities (top panel) peak in controls at layer 4. The density of radially oriented vessels (middle panel) peaks in layers 1,2 and 3 while decreasing in the layers 4, 5 and 6. (G) 7 days post-infection brain. Expression of the N viral protein by neural cells are shown at the level of the nose somatosensory cortex (300μm projection plane and mapped densities). While branch point densities of vessels still show a peak in layer 4, the normal radial organization of the vessels is not measured in the Nose region (arrowhead). Representative render of the vascular graph are showing a decrease in the vessels orientations seen in the control in layers 2 and 3.(H) Schematic of experiment for (I-J), adeno-associated virus coding for human ACE2 (AAV-hACE2) were injected in to the cisterna magna or intratracheally to induce brain specific or lung specific expression of hACE2. Brain hACE2 expressing mice were infected with SARS-CoV-2 intraventricularly, and lung hACE2 expressing mice were infected with SARS-CoV-2 intranasally. (I) Weight loss curve and (J) survival curve of mice infected with SARS-CoV-2 in the lung (blue) and the brain (red and orange) (blue, n=10; red, n=4; orange, n=4). Scale bars are 1mm (A,C), 200μm (B) and 500μm (D,E) Abbreviations: CB: Cerebellum, DCN: Deep Cerebellar Nuclei, GPi: Globus Pallidus internal segment, IC: Inferior Colliculus, SC: Superior Colliculus, SN: Substantia Nigra (reticulata or compacta), SS: Somatosensory Cortex, Nose of Barrel FielD, VMH: Ventro Medial Hypothalamus
Figure 5:. Evidence of neuroinvasion in post-mortem…
Figure 5:. Evidence of neuroinvasion in post-mortem COVID-19 patient brains.
FFPE sections of brain tissue from COVID-19 patients were stained using H&E and anti-SARS-CoV-2-spike antibody. (A) Image of cortical neurons positive for SARS-CoV-2 (black arrows). (B) Images of unaffected regions (left) and infected regions (right) demonstrating infection of neurons (top row) and microvasculature (bottom row). (C) Ischemic infarcts found at different stages stained with H&E (top row) and SARS-CoV-2-spike antibody (bottom row). (D) Ischemic region outlined with dotted line with positive staining focused around ischemic infarct. Bottom image shows zoomed in image indicated by dotted box in top image, and black arrows indicate infected neurons in the region.

References

    1. Amin N.D., and Pasca S.P. (2018). Building Models of Brain Disorders with Three-Dimensional Organoids. Neuron 100, 389–405.
    1. Baig A.M., and Sanders E.C. (2020). Potential Neuroinvasive Pathways of SARS-CoV-2: Deciphering the Spectrum of Neurological Deficit Seen in Coronavirus Disease 2019 (COVID-19). J Med Virol.
    1. Blanco-Melo D., Nilsson-Payant B.E., Liu W.C., Uhl S., Hoagland D., Moller R., Jordan T.X., Oishi K., Panis M., Sachs D., et al. (2020). Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. Cell 181, 1036–1045 e1039.
    1. Cakir B., Xiang Y., Tanaka Y., Kural M.H., Parent M., Kang Y.J., Chapeton K., Patterson B., Yuan Y., He C.S., et al. (2019). Engineering of human brain organoids with a functional vascular-like system. Nat Methods 16, 1169–1175.
    1. Cantuti-Castelvetri L., Ojha R., Pedro L.D., Djannatian M., Franz J., Kuivanen S., Kallio K., Kaya T., Anastasina M., Smura T., et al. (2020). Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system. bioRxiv.
    1. Coolen T., Lolli V., Sadeghi N., Rovai A., Trotta N., Taccone F.S., Creteur J., Henrard S., Goffard J.C., De Witte O., et al. (2020). Early postmortem brain MRI findings in COVID-19 non-survivors. Neurology.
    1. Daly J.L., Simonetti B., Antón-Plágaro C., Kavanagh Williamson M., Shoemark D.K., Simón-Gracia L., Klein K., Bauer M., Hollandi R., Greber U.F., et al. (2020). Neuropilin-1 is a host factor for SARS-CoV-2 infection. bioRxiv.
    1. Daniels B.P., Kofman S.B., Smith J.R., Norris G.T., Snyder A.G., Kolb J.P., Gao X., Locasale J.W., Martinez J., Gale M. Jr., et al. (2019). The Nucleotide Sensor ZBP1 and Kinase RIPK3 Induce the Enzyme IRG1 to Promote an Antiviral Metabolic State in Neurons. Immunity 50, 64–76 e64.
    1. De Felice F.G., Tovar-Moll F., Moll J., Munoz D.P., and Ferreira S.T. (2020). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the Central Nervous System. Trends Neurosci 43, 355–357.
    1. Garcez P.P., Loiola E.C., Madeiro da Costa R., Higa L.M., Trindade P., Delvecchio R., Nascimento J.M., Brindeiro R., Tanuri A., and Rehen S.K. (2016). Zika virus impairs growth in human neurospheres and brain organoids. Science 352, 816–818.
    1. Heneka M.T., Golenbock D., Latz E., Morgan D., and Brown R. (2020). Immediate and long-term consequences of COVID-19 infections for the development of neurological disease. Alzheimers Res Ther 12, 69.
    1. Hoffmann M., Kleine-Weber H., Schroeder S., Kruger N., Herrler T., Erichsen S., Schiergens T.S., Herrler G., Wu N.H., Nitsche A., et al. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271–280 e278.
    1. Hosier H., Farhadian S.F., Morotti R.A., Deshmukh U., Lu-Culligan A., Campbell K.H., Yasumoto Y., Vogels C.B., Casanovas-Massana A., Vijayakumar P., et al. (2020). SARS-CoV-2 infection of the placenta. J Clin Invest.
    1. Israelow B., Song E., Mao T., Lu P., Meir A., Liu F., Madel Alfajaro M., Wei J., Dong H., Homer R.J., et al. (2020). Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. bioRxiv.
    1. Kanton S., Boyle M.J., He Z., Santel M., Weigert A., Sanchis-Calleja F., Guijarro P., Sidow L., Fleck J.S., Han D., et al. (2019). Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418–422.
    1. Lancaster M.A., and Knoblich J.A. (2014). Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc 9, 2329–2340.
    1. Lancaster M.A., Renner M., Martin C.A., Wenzel D., Bicknell L.S., Hurles M.E., Homfray T., Penninger J.M., Jackson A.P., and Knoblich J.A. (2013). Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379.
    1. Li M.Y., Li L., Zhang Y., and Wang X.S. (2020). Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infect Dis Poverty 9, 45.
    1. Mao L., Jin H., Wang M., Hu Y., Chen S., He Q., Chang J., Hong C., Zhou Y., Wang D., et al. (2020). Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China. JAMA Neurol.
    1. McCray P.B. Jr., Pewe L., Wohlford-Lenane C., Hickey M., Manzel L., Shi L., Netland J., Jia H.P., Halabi C., Sigmund C.D., et al. (2007). Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol 81, 813–821.
    1. Netland J., Meyerholz D.K., Moore S., Cassell M., and Perlman S. (2008). Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J Virol 82, 7264–7275.
    1. Pellegrini L., Bonfio C., Chadwick J., Begum F., Skehel M., and Lancaster M.A. (2020). Human CNS barrier-forming organoids with cerebrospinal fluid production. Science.
    1. Pereira A. (2020). Long-Term Neurological Threats of COVID-19: A Call to Update the Thinking About the Outcomes of the Coronavirus Pandemic. Front Neurol 11, 308.
    1. Puelles V.G., Lutgehetmann M., Lindenmeyer M.T., Sperhake J.P., Wong M.N., Allweiss L., Chilla S., Heinemann A., Wanner N., Liu S., et al. (2020). Multiorgan and Renal Tropism of SARS-CoV-2. N Engl J Med.
    1. Qi F., Qian S., Zhang S., and Zhang Z. (2020). Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses. Biochem Biophys Res Commun 526, 135–140.
    1. Qian X., Nguyen H.N., Song M.M., Hadiono C., Ogden S.C., Hammack C., Yao B., Hamersky G.R., Jacob F., Zhong C., et al. (2016). Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell 165, 1238–1254.
    1. Ramani A., Müller L., Ostermann P.N., Gabriel E., Abida-Islam P., Müller-Schiffmann A., Mariappan A., Goureau O., Gruell H., Walker A., et al. (2020). SARS-CoV-2 targets cortical neurons of 3D human brain organoids and shows neurodegeneration-like effects. bioRxiv.
    1. Renier N., Adams E.L., Kirst C., Wu Z., Azevedo R., Kohl J., Autry A.E., Kadiri L., Umadevi Venkataraju K., Zhou Y., et al. (2016). Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes. Cell 165, 1789–1802.
    1. Renier N., Wu Z., Simon D.J., Yang J., Ariel P., and Tessier-Lavigne M. (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910.
    1. Roberts A., Deming D., Paddock C.D., Cheng A., Yount B., Vogel L., Herman B.D., Sheahan T., Heise M., Genrich G.L., et al. (2007). A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog 3, e5.
    1. Solomon I.H., Normandin E., Bhattacharyya S., Mukerji S.S., Keller K., Ali A.S., Adams G., Hornick J.L., Padera R.F. Jr., and Sabeti P. (2020). Neuropathological Features of Covid-19. N Engl J Med.
    1. Sun S.H., Chen Q., Gu H.J., Yang G., Wang Y.X., Huang X.Y., Liu S.S., Zhang N.N., Li X.F., Xiong R., et al. (2020). A Mouse Model of SARS-CoV-2 Infection and Pathogenesis. Cell Host Microbe.
    1. Sungnak W., Huang N., Becavin C., Berg M., Queen R., Litvinukova M., Talavera-Lopez C., Maatz H., Reichart D., Sampaziotis F., et al. (2020). SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat Med 26, 681–687.
    1. Velasco S., Kedaigle A.J., Simmons S.K., Nash A., Rocha M., Quadrato G., Paulsen B., Nguyen L., Adiconis X., Regev A., et al. (2019). Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570, 523–527.
    1. Watanabe M., Buth J.E., Vishlaghi N., de la Torre-Ubieta L., Taxidis J., Khakh B.S., Coppola G., Pearson C.A., Yamauchi K., Gong D., et al. (2017). Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection. Cell Rep 21, 517–532.
    1. Xia H., and Lazartigues E. (2008). Angiotensin-converting enzyme 2 in the brain: properties and future directions. J Neurochem 107, 1482–1494.
    1. Xu Y.P., Qiu Y., Zhang B., Chen G., Chen Q., Wang M., Mo F., Xu J., Wu J., Zhang R.R., et al. (2019). Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids. Cell Res 29, 265–273.
    1. Yordy B., Iijima N., Huttner A., Leib D., and Iwasaki A. (2012). A neuron-specific role for autophagy in antiviral defense against herpes simplex virus. Cell Host Microbe 12, 334–345.
    1. Zhang P., Li J., Liu H., Han N., Ju J., Kou Y., Chen L., Jiang M., Pan F., Zheng Y., et al. (2020). Long-term bone and lung consequences associated with hospital-acquired severe acute respiratory syndrome: a 15-year follow-up from a prospective cohort study. Bone Res 8, 8.

Source: PubMed

Спонсоры и соавторы

Заболевания

Медикаментозные вмешательства

Подписаться