Part 2 of a two-part series. Click here for Part 1.
It turns out a person’s risk associated with ApoE4 goes beyond Alzheimer’s and vascular disease. This apolipoprotein allele can also worsen infections caused by certain viruses. One of those appears to be SARS-CoV-2, the cause of COVID-19, according to two epidemiological studies. In the January 11 Gerontology, researchers led by Miguel Calero, Queen Sofia Foundation Alzheimer Research Center, Madrid, Spain, reported that aged ApoE4 carriers are more likely to show COVID-19 symptoms than ApoE3 carriers. This supports a paper in the September 16, 2020, Journals of Gerontology. Researchers led by David Melzer, University of Exeter, England, U.K., reported that older ApoE4 carriers are more likely to test positive for the virus and more likely to die from COVID-19 than are ApoE3s carriers. Why might this be?
- People who carry two copies of ApoE4 are more likely to get infected and die with COVID-19.
- Astrocyte-neuron co-culture boosts viral infection of each cell.
- Infected ApoE4 neurons have shorter neurites, fewer synapses, than E3s.
In the January 4 Cell Stem Cell, researchers led by Yanhong Shi, Beckman Research Institute, Duarte, California, and Vaithilingaraja Arumugaswami, University of California, Los Angeles, reported that the new coronavirus infected more ApoE4 neurons and astrocytes than their ApoE3 counterparts in cell culture. Astrocytes stoked the fire, upping the number of infected cells in co-cultures and in astrocyte-containing brain organoids. Infected neurons degenerated, while astrocytes swelled and their nuclei broke apart. Though this may not explain why ApoE4 carriers are at higher risk of COVID-19, it suggests that they may be more prone to long-term neurological symptoms of the disease (see Part 1 of this series).
To probe how various factors, including ApoE genotype, affect the severity of a person’s COVID-19, Calero and colleagues phoned people ages 75-94 who, as part of in the Vallecas Project in Madrid, had been genotyped for ApoE. From 2011–2013, this observational cohort study tracked markers that might predict future dementia (Olarzaran et al., 2015). In April 2020, first author Teodoro del Ser Claero asked Vallecas participants if they had any COVID-19 symptoms, and learned that ApoE4 carriers were 2.4 times as likely to reply in the affirmative, and to have had a COVID-19 diagnosis.
This aligns with Melzer and colleagues’ data. They sifted through the U.K. Biobank database, which now includes COVID-19 infection data. First author Chia-Ling Kuo stratified participants based on APOE genotype. ApoE4/4 carriers were 2.2 times as likely to have tested positive or have had severe disease, and 4.3 times as likely to have died from COVID-19 than were ApoE3/3s. These differences remained even after the researchers corrected for comorbidities known to worsen COVID-19, such as dementia, hypertension, and Type 2 diabetes.
Even so, Caleb Finch, University of Southern California, Los Angeles, and Alexander Kulminski, Duke University, Durham, North Carolina, think the comorbidities may explain the association. “ApoE cluster haplotypes associate with the same morbidities from cardiovascular disease and obesity that increase vulnerability to COVID-19,” they note in a review in the same journal. The ApoE locus was first recognized as a genetic determinant of cardiovascular disease in the 1980s, through its effect on blood lipid and cholesterol levels (Sing et al., 1985). Some research even indicated ApoE4 protected people from lipophilic pathogens (Martin, 1999). But at least for COVID, the latest data suggest the opposite. “The ApoE trail, like a Moebius strip, takes us back to where we started from, four decades ago, with another view,” wrote Finch and Kulminski. “To understand how ApoE4 may increase COVID-19 infectivity and mortality, we have returned to the original associations of ApoE variants with blood lipids, vascular disease, and cognition.”
Zooming in to the cellular level may provide insight on how ApoE4 renders cells more susceptible to viruses. For example, HIV more easily penetrates human cells if they are ApoE4/4 (Burt et al., 2008). In mice, herpes simplex virus (HSV) exploits the lipoprotein to enter brain cells, leading to a higher viral burden in ApoE4 than ApoE3 transgenic mice (Burgos et al., 2006). What about SARS-CoV-2?
ApoE4 Worsens SARS-CoV-2 Damage. The virus infected neurons and astrocytes in cell culture and brain organoids. ApoE4 cells fared worse, remdesivir protected them. [Courtesy of Wang et al., Cell Stem Cell, 2021.]
Shi wondered if ApoE4 could explain the neurological effects of SARS-CoV-2 in some people. To begin with, co-first authors Cheng Wang, Mingzi Zhang, and colleagues confirmed the virus’ penchant for certain brain cells (see Part 1 of this series). They differentiated human induced pluripotent stem cells (hiPSCs) into neuronal progenitors (NPCs), neurons, astrocytes, oligodendrocyte progenitor, or brain endothelial cells, then infected them with SARS-CoV-2. Immunostaining detected the viral spike protein in less than 5 percent of NPCs, neurons, and astrocytes, which Shi called a low-grade infection. The virus also infected 60-day-old brain organoids comprising NPCs and neurons.
Astrocytes are known to spread neurotropic viruses in the CNS, including Japanese encephalitis, West Nile, and Zika viruses (Soung et al., 2018; Potokar et al., 2019). Could astrocytes stoke the COVID fire in neurons and organoids? Indeed, more neurons tested positive for SARS-CoV-2 spike protein in neuron-astrocyte co-cultures than in monoculture. The scientists also saw higher viral RNA loads in neurons from organoids that had incorporated astrocytes than in those that did not.
Wang, Zhang, and colleagues then homed in on APOE genotype. They used CRISPR/Cas9 to create isogenic cell lines from iPSCs taken from ApoE3/3 and ApoE4/4 donors. They differentiated the cells into neurons and co-cultured them with ApoE3 astrocytes for three weeks. Then they added SARS-CoV-2, using one viral particle per cell. Immunostaining revealed spike protein in all neurons within 24 hours. After 72 hours, the viral protein content in all neurons had grown, but E4 neurons had 1.5 times more than E3 neurons (see image below).
Viral Invasion. After SARS-Cov-2 infects neurons (purple, left panels), its spike protein (green) popped up within 24 hours (middle) and accumulated over 72 hours (right panel). Infected ApoE4 neurons (bottom) had more viral protein than isogenic ApoE3 lines (top). [Courtesy of Wang et al., Cell Stem Cell, 2021.]
At that point, infected cells formed fewer neurites than did uninfected cells, and the neurites were short. Infected ApoE4 cells had even fewer neurites than ApoE3 cells, and they were shorter still. Staining with Syn 1 revealed fewer synapses in both infected neurons.
What about astrocytes? More iPSC-derived ApoE4/4 astrocytes were infected than iPSC-derived ApoE3/3 cells. The former had fatter somas, longer processes, and nuclei that were more fragmented compared to infected ApoE3 cells (see image below). Taken together, these findings hint at an ApoE-dependent reaction to viral infection, with ApoE4 neurons and astrocytes more severely damaged.
Angrier Astrocytes. SARS-CoV-2-infected (red) E4 astrocyte soma (right four panels) had fragmented nuclei (blue) and grew fatter (green) than E3 astrocyte soma (left four panels) whose nuclei remained intact. [Courtesy of Wang et al., Cell Stem Cell, 2021.]
Why were E4 astrocytes worse off than the E3 cells? Jessica Young, University of Washington, Seattle, thinks it may have to do with endosomes. Of 40 genes previously identified as crucial for SARS-CoV-2 infection, two, the endosomal entry receptor ACTR2 and the ATP6AP2 ATPase, are involved in endosome function (Daniloski et al., 2021). Both are more highly expressed in ApoE4 astrocytes, which have larger early endosomes than do E3 cells (Oct 2020 news). “Proteins involved in endosomal entry and transport are more abundant in APOE4 cells, which may facilitate the cellular infectivity of the virus,” Young told Alzforum (full comment below). G. William Rebeck, Georgetown University, Washington, D.C., agreed the endosome might be involved. “The speculation that these ApoE effects may be due to differentially expressed genes related to endosomal trafficking builds on a model that has been developed across several labs over the past two decades.” (Full comment below.)
The FDA-approved drug remdesivir quelled SARS-CoV-2 infection in cultured neurons and astrocytes. When Wang, Zhang, and colleagues pretreated cells with 10 μM remdesivir two hours before adding the virus, fewer neurons and astrocytes became infected. The drug also bumped up the number and length of neurites and reduced the number of fragmented nuclei in infected astrocytes compared to vehicle treatment.
Whether this remdesivir is relevant in the clinic remain to be seen. “Remdesivir is thought to poorly enter the brain,” Rik van der Kant, Vrije Universiteit Amsterdam, and Diederik van de Beek, Amsterdam UMC, wrote in a joint comment (below). David Clifford, Washington University, St. Louis, cautioned against overinterpreting these results based on what he hears from fellow clinicians who are treating COVID-19 patients. “Clinically, remdesivir appears minimally effective and is increasingly considered unimportant in COVID-19 patient care,” he wrote. “Treating the CNS is always more challenging than treating peripheral infections.”—Chelsea Weidman Burke
- How Does COVID-19 Affect the Brain?
- In Astrocytes, ApoE4 Bungles Endocytosis, PICALM Picks Up the Slack
- Olazarán J, Valentí M, Frades B, Zea-Sevilla MA, Ávila-Villanueva M, Fernández-Blázquez MÁ, Calero M, Dobato JL, Hernández-Tamames JA, León-Salas B, Agüera-Ortiz L, López-Álvarez J, Larrañaga P, Bielza C, Álvarez-Linera J, Martínez-Martín P. The Vallecas Project: A Cohort to Identify Early Markers and Mechanisms of Alzheimer's Disease. Front Aging Neurosci. 2015;7:181. Epub 2015 Sep 30 PubMed.
- Sing CF, Davignon J. Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am J Hum Genet. 1985 Mar;37(2):268-85. PubMed.
- Martin GM. APOE alleles and lipophylic pathogens. Neurobiol Aging. 1999 Jul-Aug;20(4):441-3. 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.
- Burgos JS, Ramirez C, Sastre I, Valdivieso F. Effect of apolipoprotein E on the cerebral load of latent herpes simplex virus type 1 DNA. J Virol. 2006 Jun;80(11):5383-7. PubMed.
- Soung A, Klein RS. Viral Encephalitis and Neurologic Diseases: Focus on Astrocytes. Trends Mol Med. 2018 Nov;24(11):950-962. Epub 2018 Oct 9 PubMed.
- Potokar M, Jorgačevski J, Zorec R. Astrocytes in Flavivirus Infections. Int J Mol Sci. 2019 Feb 6;20(3) PubMed.
- Daniloski Z, Jordan TX, Wessels HH, Hoagland DA, Kasela S, Legut M, Maniatis S, Mimitou EP, Lu L, Geller E, Danziger O, Rosenberg BR, Phatnani H, Smibert P, Lappalainen T, tenOever BR, Sanjana NE. Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells. Cell. 2021 Jan 7;184(1):92-105.e16. Epub 2020 Oct 24 PubMed.
- Inal J. Biological Factors Linking ApoE ε4 Variant and Severe COVID-19. Curr Atheroscler Rep. 2020 Oct 2;22(11):70. PubMed.
- Moriarty PM, Gorby LK, Stroes ES, Kastelein JP, Davidson M, Tsimikas S. Lipoprotein(a) and Its Potential Association with Thrombosis and Inflammation in COVID-19: a Testable Hypothesis. Curr Atheroscler Rep. 2020 Jul 25;22(9):48. PubMed.
- Rahman MA, Islam K, Rahman S, Alamin M. Neurobiochemical Cross-talk Between COVID-19 and Alzheimer's Disease. Mol Neurobiol. 2021 Mar;58(3):1017-1023. Epub 2020 Oct 19 PubMed.
- Fotuhi M, Mian A, Meysami S, Raji CA. Neurobiology of COVID-19. J Alzheimers Dis. 2020;76(1):3-19. 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:S183-8. PubMed.
- 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.
- Del Ser T, Fernández-Blázquez MA, Valentí M, Zea-Sevilla MA, Frades B, Alfayate E, Saiz L, Calero O, García-López FJ, Rábano A, Medina M, Calero M. Residence, Clinical Features, and Genetic Risk Factors Associated with Symptoms of COVID-19 in a Cohort of Older People in Madrid. Gerontology. 2021;67(3):281-289. Epub 2021 Jan 11 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.
- Finch CE, Kulminski AM. The ApoE Locus and COVID-19: Are We Going Where We Have Been?. J Gerontol A Biol Sci Med Sci. 2021 Jan 18;76(2):e1-e3. PubMed.