Wang C, Zhang M, Garcia G Jr, Tian E, Cui Q, Chen X, Sun G, Wang J, Arumugaswami V, Shi Y. ApoE-Isoform-Dependent SARS-CoV-2 Neurotropism and Cellular Response. Cell Stem Cell. 2021 Feb 4;28(2):331-342.e5. Epub 2021 Jan 4 PubMed.

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  1. This is an interesting paper by Wang et al. showing that APOE4 is a genetic modifier facilitating the direct infection of SARS-CoV-2 in human iPSC-derived neurons and astrocytes. I was surprised by this. Future studies should define if APOE4 causes this phenotype through gain of toxic effects or loss of protective function.

    These findings may be linked to neurological symptoms. When SARS-CoV-2 infects neurons, diverse neurological symptoms will be induced due to neuronal damage and subsequent neuroinflammation depending on affected brain regions and neuron subtypes. Loss of smell/taste may be induced by the direct infection of neurons. However, further studies are necessary to determine if SARS-CoV-2 predominantly invades the central nervous system and sufficiently infects neurons.

    APOE4 is associated with cerebrovascular dysfunction and altered microglial activation in AD. Thus, systemic inflammation and vascular damage in COVID-19 may be aggravated in APOE4 carriers, which could possibly be linked to the pathogenesis of COVID-induced delirium. It is also important to explore how SARS-CoV-2 infection influences Aβ and tau accumulation in future studies.

    If cell surface HSPG mediates the entrance of SARS-CoV-2, APOE may influence the infectivity by modulating the HSPG-virus interaction as APOE has heparin binding regions. In addition, since lipids are major components of virus envelopes, APOE may also modulate the interaction of a virus with the cell membrane by affecting membrane lipid distributions.

    In relation to Kuo et al.’s finding that APOE4/4 carriers had worse COVID-19 outcomes (Kuo et al., 2020), it is possible that APOE4 exacerbates SARS-CoV-2 infection in peripheral tissues, including lung and blood vessels, through common mechanisms, thereby increasing the severity of COVID-19 infection.

    References:

    Kuo CL, Pilling LC, Atkins JL, Masoli JA, Delgado J, Kuchel GA, Melzer D. ApoE e4e4 Genotype and Mortality With COVID-19 in UK Biobank. J Gerontol A Biol Sci Med Sci. 2020 Sep 16;75(9):1801-1803. PubMed.

    View all comments by Takahisa Kanekiyo

  2. ApoE isoform-dependent SARS-CoV-2 neurotropism and cellular response

    The paper by Wang, Zhang, and colleagues suggests that ApoE4/4 neurons and astrocytes are more readily infected by SARS-CoV2 than ApoE3/3 neurons. This finding is interesting but the clinical impact remains unclear. Patients with COVID-19 may have co-existing neurological symptoms, such as ageusia and stroke, but SARS-CoV-2 itself is often not detected in postmortem brain tissue in patients with COVID-19 (e.g., Lee et al., 2020). A number of groups have evaluated the efficiency of SARS-CoV-2 to infected cultured (human) neurons, with mixed results.

    The current study adds a new twist to the story. An epidemiological study had described the ApoE4 genotype as a risk factor for severe COVID-19 (Kuo et al., 2020). This could indicate that an ApoE4 genotype benefits SARS-CoV2 infection and/or replication or affects the host immune-response to SARS-CoV-2 driving more severe COVID-19. However, it is doubtful that brain cells play a major role in these initial steps that lead to severe COVID-19. Whether ApoE4 is also associated with neurological symptoms, as the authors in the current study suggest in their discussion, has not been addressed yet in clinical studies. The authors also claim, based on their findings in iPSC-neurons and astrocytes, that remdesivir could be used to treat neurological complications in COVID-19 patients. However, as remdesivir is thought to poorly enter the brain, it is unclear whether this has any clincial implication. 

    We believe that the current findings give much food for thought. For example, it would be of interest to test if SARS-CoV-2 infection is also affected by ApoE4-genotype in (iPSC-derived) epithelial or alveolar cells. There are a lot of unknowns in this pandemic. So far there is no clear indication that COVID-19 enters the brain. However, if COVID-19 infects neurons and astrocytes in living humans and selectively causes neuronal death in ApoE4-genotypes, as suggested by this study, this is bad news for ApoE4/4 carriers who are already at higher risk for AD and possibly also severe COVID-19.

    Epidemiological data will be essential to further address this issue, before any far-reaching conclusions can be drawn.

    References:

    Lee MH, Perl DP, Nair G, Li W, Maric D, Murray H, Dodd SJ, Koretsky AP, Watts JA, Cheung V, Masliah E, Horkayne-Szakaly I, Jones R, Stram MN, Moncur J, Hefti M, Folkerth RD, Nath A. Microvascular Injury in the Brains of Patients with Covid-19. N Engl J Med. 2020 Dec 30; PubMed.

    Kuo CL, Pilling LC, Atkins JL, Masoli JA, Delgado J, Kuchel GA, Melzer D. ApoE e4e4 Genotype and Mortality With COVID-19 in UK Biobank. J Gerontol A Biol Sci Med Sci. 2020 Sep 16;75(9):1801-1803. PubMed.

    View all comments by Diederik van de Beek

  3. This is an interesting and timely work using hiPSC-derived neural models to understand the potential implications of SARS-CoV-2 infection in the brain. While neurological symptoms have been attributed to infection, the mechanisms behind these symptoms are not clear. This study uncovers several novel biological impacts: first showing that neurons are more highly infected in the presence of astrocytes, second, that APOE4 genotype cells have higher infectivity and stronger phenotypes, and finally that these cells may express higher levels of the receptors and machinery needed for endosomal entry and transport of viral particles.

    APOE4 is the strongest risk factor for development of Alzheimer’s disease as well, and likely plays a different role in the different CNS cell types in how that risk manifests on a cellular level. This study shows that to be the case in the face of the SARS-CoV-2 challenge. However, although this study shows that, in vitro, neurons and astrocytes are directly infected by SARS-CoV-2, in the human brain the role of the blood-brain barrier must be considered. The authors discuss this in the paper, however, it is still unclear whether the neurological phenotypes in patients are due to direct infection or due to neuroinflammation because of BBB breakdown. It will be necessary to follow COVID-19 survivors and monitor them for neurologic effects as they age.

    View all comments by Jessica Young

  4. This fascinating study by Wang et al. further expands the already numerous roles of Apolipoprotein E (APOE) in health and disease to now include the world’s major public health challenge, COVID-19. The authors employ a variety of human iPSC lines and brain organoid systems to model the infectivity and susceptibility of various CNS cell types to SARS-CoV-2. They first confirm SARS-CoV-2 infection in human iPSC-derived neurons and astrocytes, as has been reported by several groups. Likely of more interest to the Alzheimer’s disease research community are the findings of an increased rate of SARS-CoV-2 infection, and more severe cellular responses, in neurons and astrocytes expressing the ε4 allele of APOE. As the authors themselves allude to, these data provide one potential explanation for why some individuals (i.e., those carrying APOE4), but not all COVID-19 patients, show neurological manifestations.

    Notably, a study published last year identified an association between E4 carriage and COVID-19 severity (Kuo et al., 2020). Analyzing over 400,000 individuals in the U.K. Biobank, the authors observed that ε4/ε4 individuals were more likely to be COVID-19-positive compared to ε3 homozygotes (odds ratio >2.0), and that this effect was independent of known comorbidities. Presciently, an opinion written one month earlier had raised this very possibility—that E4 may predict an individual’s propensity to manifest more severe illness with COVID-19 (Goldstein et al., 2020).

    The authors based their hypothesis on several factors that parallel questions raised by this current study. First, E4 is associated with several comorbidities that put one at higher risk for severe illness with COVID-19, including dementia, atherosclerosis, and hypertension (Mahley et al., 2009; Niu et al., 2009). Second, as severe COVID-19 is characterized by acute respiratory distress syndrome (ARDS), they note that ApoE is expressed in several cell types in the lung, and that ApoE has been shown to act as a concentration-dependent activation signal in asthmatic individuals. Third, harkening back to the cytokine storm that drives ARDS, possession of E4 has been associated with an amplified innate immune response (e.g., higher cytokine levels and hyperthermia in E4+ subjects injected with LPS (Gale et al., 2014). Last, and perhaps most notably, APOE has an established role in modulating infectivity and symptomology of several common viruses, including HIV, hepatitis C virus (HCV), and herpes simplex virus 1 (reviewed by Kuhlman et al., 2010). Further, this modulatory role of APOE includes effects on cognitive processes (e.g., E4 allele status modifies the relationship between herpes simplex virus 1 (HSV-1) and total infectious burden and cognitive function (Zhao et al., 2020)). 

    So, does the pleiotropic ApoE protein simply modulate entry into the cell, or does it regulate pathways downstream of entry, including replication, trafficking, or associated damage? Although the current study does not dissect these two factors, Wang et al.’s data suggest that E4 results in both increased infection rate (higher percent of spike-protein-positive cells) and more exaggerated viral-associated neuropathology (neuritic degeneration, synapse loss, enlarged cell bodies, and syncytia formation).

    How might ApoE modulate these processes? In regard to entry, the literature may provide some clues in that both ApoE and several viruses compete for entry into the cell via heparin sulfate proteoglycans (HSPGs). Interestingly, ACE2 receptor-mediated entry of SARS-CoV-2 into a cell requires heparan sulfate as a co-factor (Zhang et al., 2020). Perhaps the increased infectivity observed in the presence of APOE4 is a result of isoform-specific differences in ApoE availability and/or binding to HSPGs.

    In regard to replication, one potential explanation may trace back to ApoE’s essential role as a lipid carrier. Lipid droplets, neutral lipid-rich intracellular organelles that regulate lipid storage and utilization, are used as a replication center for several viruses, including HCV, dengue virus, and rotavirus (Dias et al., 2020). Several groups, including our own, have described increases in lipid droplet formation and metabolism in cells expressing E4 (reviewed by Farmer et al., 2020). Perhaps these organelles—and their differential regulation by E4—provide a link to the SARS-CoV-2 dynamics in neurons and astrocytes described here by Wang et al.?

    Speaking of astrocytes, the authors show that the rate of infection was about twofold higher in iPSC-derived astrocytes than in neurons (although this reflects a change from only ~1 percent to ~2 percent spike-protein-positive cells). Also of note was that the presence of astrocytes significantly increased the infection rates of neurons, both in two-dimensional co-culture systems and in three-dimensional brain organoid models. Knowing that astrocytes (normally) secrete substantially higher levels of ApoE than neurons, this raises several questions. Are cells that synthesize more ApoE at higher risk for SARS-CoV-2 infection (i.e., does infectivity and/or cellular pathology correlate with local ApoE concentrations)?

    And what about microglia? While the study here by Wang et al. shows exciting results in astrocytes, neurons, neural progenitor cells, and oligodendrocyte progenitor cells, the jury is still out on a central player in the CNS, and a known ApoE factory (at least upon activation). There is a growing consensus that microglia-derived ApoE is central to Alzheimer’s disease pathogenesis (Keren-Shaul et al., 2017; Krasemann et al., 2017; Shi et al., 2017). So how do these brain-resident immune cells respond to SARS-CoV-2? Does the dramatic upregulation of ApoE during microglia activation play a role? Do microglial ApoE-related pathways help determine which individuals infected with COVID-19 will suffer neurological symptoms and which won’t?

    As with any interesting finding, many more questions now arise. Given the ubiquitous and persistent threat of COVID-19, and the high population frequency and dramatic AD risk associated with APOE4, answering those questions will be an important and pressing new challenge for researchers.

    References:

    Goldstein MR, Poland GA, Graeber AC. Does apolipoprotein E genotype predict COVID-19 severity?. QJM. 2020 Aug 1;113(8):529-530. PubMed.

    Burt TD, Agan BK, Marconi VC, He W, Kulkarni H, Mold JE, Cavrois M, Huang Y, Mahley RW, Dolan MJ, McCune JM, Ahuja SK. Apolipoprotein (apo) E4 enhances HIV-1 cell entry in vitro, and the APOE epsilon4/epsilon4 genotype accelerates HIV disease progression. Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8718-23. PubMed.

    Dias SS, Soares VC, Ferreira AC, Sacramento CQ, Fintelman-Rodrigues N, Temerozo JR, Teixeira L, Nunes da Silva MA, Barreto E, Mattos M, de Freitas CS, Azevedo-Quintanilha IG, Manso PP, Miranda MD, Siqueira MM, Hottz ED, Pão CR, Bou-Habib DC, Barreto-Vieira DF, Bozza FA, Souza TM, Bozza PT. Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators. PLoS Pathog. 2020 Dec;16(12):e1009127. Epub 2020 Dec 16 PubMed.

    Farmer BC, Walsh AE, Kluemper JC, Johnson LA. Lipid Droplets in Neurodegenerative Disorders. Front Neurosci. 2020;14:742. Epub 2020 Jul 29 PubMed.

    Gale SC, Gao L, Mikacenic C, Coyle SM, Rafaels N, Murray Dudenkov T, Madenspacher JH, Draper DW, Ge W, Aloor JJ, Azzam KM, Lai L, Blackshear PJ, Calvano SE, Barnes KC, Lowry SF, Corbett S, Wurfel MM, Fessler MB. APOε4 is associated with enhanced in vivo innate immune responses in human subjects. J Allergy Clin Immunol. 2014 Jul;134(1):127-34. Epub 2014 Mar 18 PubMed.

    Gordon EM, Yao X, Xu H, Karkowsky W, Kaler M, Kalchiem-Dekel O, Barochia AV, Gao M, Keeran KJ, Jeffries KR, Levine SJ. Apolipoprotein E is a concentration-dependent pulmonary danger signal that activates the NLRP3 inflammasome and IL-1β secretion by bronchoalveolar fluid macrophages from asthmatic subjects. J Allergy Clin Immunol. 2019 Aug;144(2):426-441.e3. Epub 2019 Mar 11 PubMed.

    Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8 PubMed.

    Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O'Loughlin E, Xu Y, Fanek Z, Greco DJ, Smith ST, Tweet G, Humulock Z, Zrzavy T, Conde-Sanroman P, Gacias M, Weng Z, Chen H, Tjon E, Mazaheri F, Hartmann K, Madi A, Ulrich JD, Glatzel M, Worthmann A, Heeren J, Budnik B, Lemere C, Ikezu T, Heppner FL, Litvak V, Holtzman DM, Lassmann H, Weiner HL, Ochando J, Haass C, Butovsky O. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    Kuhlmann I, Minihane AM, Huebbe P, Nebel A, Rimbach G. Apolipoprotein E genotype and hepatitis C, HIV and herpes simplex disease risk: a literature review. Lipids Health Dis. 2010;9:8. PubMed.

    Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS. J Lipid Res. 2009 Apr;50 Suppl(Suppl):S183-8. Epub 2008 Dec 22 PubMed.

    Niu W, Qi Y, Qian Y, Gao P, Zhu D. The relationship between apolipoprotein E epsilon2/epsilon3/epsilon4 polymorphisms and hypertension: a meta-analysis of six studies comprising 1812 cases and 1762 controls. Hypertens Res. 2009 Dec;32(12):1060-6. Epub 2009 Oct 9 PubMed.

    Shi Y, Yamada K, Liddelow SA, Smith ST, Zhao L, Luo W, Tsai RM, Spina S, Grinberg LT, Rojas JC, Gallardo G, Wang K, Roh J, Robinson G, Finn MB, Jiang H, Sullivan PM, Baufeld C, Wood MW, Sutphen C, McCue L, Xiong C, Del-Aguila JL, Morris JC, Cruchaga C, Alzheimer’s Disease Neuroimaging Initiative, Fagan AM, Miller BL, Boxer AL, Seeley WW, Butovsky O, Barres BA, Paul SM, Holtzman DM. ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy. Nature. 2017 Sep 28;549(7673):523-527. Epub 2017 Sep 20 PubMed.

    Shi Y, Manis M, Long J, Wang K, Sullivan PM, Remolina Serrano J, Hoyle R, Holtzman DM. Microglia drive APOE-dependent neurodegeneration in a tauopathy mouse model. J Exp Med. 2019 Nov 4;216(11):2546-2561. Epub 2019 Oct 10 PubMed.

    Zhang Q, Chen CZ, Swaroop M, Xu M, Wang L, Lee J, Wang AQ, Pradhan M, Hagen N, Chen L, Shen M, Luo Z, Xu X, Xu Y, Huang W, Zheng W, Ye Y. Targeting heparan sulfate proteoglycan-assisted endocytosis as a COVID-19 therapeutic option. bioRxiv. 2020 Jul 14; PubMed.

    Zhao C, Strobino K, Moon YP, Cheung YK, Sacco RL, Stern Y, Elkind MS. APOE ϵ4 modifies the relationship between infectious burden and poor cognition. Neurol Genet. 2020 Aug;6(4):e462. Epub 2020 Jul 7 PubMed.

    View all comments by Lance Johnson

  5. Our findings show that the SARS-CoV-2 viral spike protein S1, which is required for cellular uptake of virus, can enter the mouse brain following intravenous or intranasal administration. This might relate to the fact that people with COVID-19 are often suffering cognitive effects, such as brain fog, and fatigue.

    This finding is also important because binding proteins such as S1 can detach from the virus and themselves cause toxicity in patients following COVID-19 infection. The S1 protein in SARS-CoV-2 and the gp 120 protein in HIV-1 might function similarly. They are glycoproteins—proteins that have a lot of sugars on them, hallmarks of proteins that bind to other receptors. Both proteins function as the arms and hands for their viruses by grabbing onto other glycoproteins on the cell surface. Both cross the blood-brain barrier and S1, like gp120, is likely toxic to brain tissues. We (Bill Banks and myself) worked with recombinant gp 120 as part of previous mouse studies.

    The breathing problems seen following COVID-19 infection might not only relate to infection in the lungs but also to respiratory centers of the brain following viral uptake in the brain. We find that transport of S1 is faster in the olfactory bulb and kidney of males than females. This observation might relate to the increased susceptibility of men to more severe COVID-19 outcomes. Our findings that wild-type mice, which do not express the human ACE2 receptor, can be used in kinetics studies of the S1 protein and probably SARS-CoV-2 suggest that the assumption that human ACE2 must be used for these kind of studies in mice, is incorrect. These results also support the use of recombinant proteins or virus-like proteins to increase our knowledge about the effects of SARS-CoV-2 proteins on the brain, rather than active replicating viruses, which require additional biosafety precautions and animal facilities.

    References:

    Raber J, Toggas SM, Lee S, Bloom FE, Epstein CJ, Mucke L. Central nervous system expression of HIV-1 Gp120 activates the hypothalamic-pituitary-adrenal axis: evidence for involvement of NMDA receptors and nitric oxide synthase. Virology. 1996 Dec 15;226(2):362-73. PubMed.

    Banks WA, Kastin AJ, Akerstrom V. HIV-1 protein gp120 crosses the blood-brain barrier: role of adsorptive endocytosis. Life Sci. 1997;61(9):PL119-25. PubMed.

    View all comments by Jacob Raber

  6. These findings are quite consistent with reports, espoused by many eminent researchers, and central to our stance on www.ALZgerm.org, that many viruses, bacteria, and parasites can enter the brain, either temporarily or long-term. They may live quietly, trigger inflammation, or directly damage brain cells. And so, COVID joins herpes simplex, spirochetes, toxoplasma, chlamydia, mycobacteria, and prions as a possible future trigger of AD.

    We also recall that after chickenpox, the neurotropic herpes zoster virus becomes latent for decades until emerging as shingles. The neurotrophic spirochetes of syphilis, and perhaps borrelia, may damage the brain years after initial infection and then latency. As I warned in comments on Alzforum April 1, 2020, COVID infections may disturb, visibly or not, future neurologic findings and outcomes in cohorts of seniors being followed longitudinally in dementia studies.

    View all comments by Leslie Norins

  7. These studies did not examine brain capillary pericytes, which, unlike endothelial cells or other perivascular cell types, express ACE2, the receptor for SARS-CoV-2 (He et al., 2020). Within pericytes of APOE4 carriers, APOE and NFAT are selectively dysregulated (Blanchard et al., 2020), and APOE4 leads to accelerated breakdown of the blood-brain barrier which is in part maintained by brain capillary pericytes (Montagne et al., 2020). Infection or dysfunction of these pericytes might cause white matter disruptions in addition to various brain vasculature problems.

    References:

    He L, Mäe MA, Muhl L, Sun Y, Pietilä R, Nahar K, Vázquez Liébanas E, Fagerlund MJ, Oldner A, Liu J, Genové G, Zhang L, Xie Y, Leptidis S, Mocci G, Stritt S, Osman A, Anisimov A, Hemanthakumar KA, Räsänen R, Mirabeau O, Hansson E, Björkegren J, Vanlandewijck M, Blomgren K, Mäkinen T, Peng XR, Arnold TD, Alitalo K, Lendahl U, Betsholtz C. Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 – implications for microvascular inflammation and hypercoagulopathy in COVID-19. bioRxiv, July 26, 2020. bioRxiv.

    Blanchard JW, Bula M, Davila-Velderrain J, Akay LA, Zhu L, Frank A, Victor MB, Bonner JM, Mathys H, Lin YT, Ko T, Bennett DA, Cam HP, Kellis M, Tsai LH. Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes. Nat Med. 2020 Jun;26(6):952-963. Epub 2020 Jun 8 PubMed. Correction.

    Montagne A, Nation DA, Sagare AP, Barisano G, Sweeney MD, Chakhoyan A, Pachicano M, Joe E, Nelson AR, D'Orazio LM, Buennagel DP, Harrington MG, Benzinger TL, Fagan AM, Ringman JM, Schneider LS, Morris JC, Reiman EM, Caselli RJ, Chui HC, Tcw J, Chen Y, Pa J, Conti PS, Law M, Toga AW, Zlokovic BV. APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature. 2020 May;581(7806):71-76. Epub 2020 Apr 29 PubMed.

    View all comments by Charles Stromeyer

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