key: cord-1026234-ee1yyg1v
authors: Ramani, Anand; Islam, Pranty Abida; Gopalakrishnan, Jay
title: Neurotropic effects of SARS-CoV-2 modeled by the human brain organoids
date: 2021-02-12
journal: Stem Cell Reports
DOI: 10.1016/j.stemcr.2021.02.007
sha: 2ccce45518c11e28c1aabeebf900cb17c25bef53
doc_id: 1026234
cord_uid: ee1yyg1v

COVID-19 caused by SARS-CoV-2 is a socioeconomic burden exhibiting respiratory illness along with unexpected neurological complications. This raised concerns about whether the observed neurological symptoms are due to direct effects on CNS or associated with the virus's systemic effect. Recent SARS-CoV-2 infection studies using human brain organoids revealed that SARS-CoV-2 targets human neurons. Human brain organoids are stem cell-derived reductionist experimental systems that have highlighted the neurotropic effects of SARS-CoV-2. Here, we summarize the neurotoxic effects of SARS-CoV-2 using brain organoids and comprehensively discuss how brain organoids could further improve our understanding when they are fine-tuned.

immunity on the function of the blood-brain barrier, which activates the CNS immune pathway and leads to the disruption of neural circuits (Najjar et al., 2020) . Further studies have discussed the potential of SARS-CoV-2 to target the respiratory centers in the brain, leading to massive inflammation and probable respiratory failure. Therefore, respiratory insufficiency is not merely associated with lung failure but could also result from the presence of interleukins, tumor necrosis factors, and other cytokines that impair the functioning of the medullary cardiorespiratory center in the brain Steardo et al., 2020) .

Intriguingly, previous studies show the capacity of SARS-CoV-2 but not SARS-CoV to replicate in U251 neuronal cells, highlighting the neuroinvasive potential of the virus . Thus, at this point, it became essential to test whether SARS-CoV-2 infects human neurons and productively replicates in the CNS. Indeed, experiments exposing SARS-CoV-2 to human brain organoids have revealed the presence of virus in neurons and other neuronal cell types (Jacob et al., 2020) (Pellegrini et al., 2020a) (Pinar Mesci, 2020; Song et al., 2020a) .

Remarkably, analyzing the brain autopsy samples of diseased COVID-19 patients, the Iwasaki lab has identified the presence of SARS-CoV-2 in the cortical neurons. This work has established a direct neuroinvasive capacity of SARS-CoV-2 in the human brain and substantiates the findings revealed by the human brain organoids. Together, thanks to human brain organoids, which allowed several groups to independently test the SARS-CoV-2's neurotropism amid this pandemic. Here, we summarize the neurotoxic effects of SARS-CoV-2 revealed by the human brain organoids. We also attempt to highlight the roadblocks of using human brain organoids and discuss how solving those limitations can help efficient modeling of COVID-19 and identify potential therapeutic agents against SARS-CoV-2.

Brain organoids are innovative experimental model systems generated from either embryonic stem cells (ES) or induced pluripotent stem cells (iPSCs). With the combination of modern 3D cultures and directed differentiation methods, the Sasai and Vaccarino laboratories are the first to exploit the self-assembling properties of pluripotent stem cells and to generate 3D neural epithelial tissues (Eiraku et al., 2008; Kadoshima et al., 2013; Mariani et al., 2012; Mariani and Vaccarino, 2019; Nakano et al., 2012) . When these neural epithelial tissues are cultured in spinner flasks or suspension cultures with defined media, they underwent self-organization to mimic in vivo tissue counterparts. Lancaster and colleagues named these objects as 3D brain organoids as they mirror many aspects of neural epithelial tissues cytoarchitecturally, similar to the developing human brain (Renner et al., 2017) (Lancaster et al., 2013) . These organoids constitute vast diversities of cell types ranging from polarized radial glia, intermediate progenitors, and layer-specific cortical J o u r n a l P r e -p r o o f neurons (Quadrato et al., 2017) (Birey et al., 2017) Pasca et al., 2015) ( Gabriel et al., 2016) .

Further advancements have generated region-specific brain organoids such as midbrain, hypothalamus, cerebellum, brain organoids with light sensitive-cell types, vasculatures mimicking the blood-brain barrier and brain organoids with microglia (Cakir et al., 2019; Li et al., 2018; Monzel et al., 2017; Ormel et al., 2018; Pellegrini et al., 2020b; Qian et al., 2018; Silva et al., 2020) .

Several elegant review articles have summarized the historical development of brain organoid cultures and various revolutionary methods, which fine-tuned brain organoid cultures suitable for detailed questions that cannot be faithfully addressed in vivo model Gopalakrishnan, 2019; Velasco et al., 2020) . For instance, early brain organoids applicable to model neurodevelopmental disorders and mature organoids appropriate to model neurodegeneration-like effects (Gabriel et al., 2016; Grenier et al., 2020; Pavoni et al., 2018) .

Elaborating the in-depth detail is out of this article's scope, and thus, we focus on how human brain organoids have helped us underpinning mechanisms of neurotropic viral infections and, in particular, SARS-CoV-2, the current strain of interest.

Most probably, the brain organoids have earned their significant momentum while modeling the disease mechanisms of Zika virus (ZIKV) during the ZIKV epidemic. Microcephaly is a rare neurodevelopmental genetic disorder caused by mutations in centrosomal and cilia genes.

However, there was a sudden rise in microcephaly infants during the ZIKV epidemic associated with their infected mothers. While ZIKV-induced microcephaly mechanisms remained a mystery, brain organoids have immensely helped identify their target cell types, consequences, disease mechanisms, and potential therapeutic agents (Gabriel et al., 2017; Qian et al., 2017; Watanabe et al., 2017) . Likewise, studies have just started to appreciate brain organoids in understanding the mechanisms of Human Cytomegalo Virus (HCMV) and Herpes Simplex Virus (HSV), neurotropic viruses to which the CNS is vulnerable. In summary, all of these works have set up a stage that human brain organoids are reliable model systems to study the infection mechanisms, target cell types, and neurotoxic effects of SARS-CoV-2. Human brain organoids are particularly instrumental to model COVID-19 because rodents have significant limitations to faithfully recapitulate human COVID-19 symptoms as they require overexpression of human ACE2 facilitating the viral entry and to exhibit the COVID-19 phenotypes (Hoffmann et al., 2020; Winkler et al., 2020; Yang et al., 2007) (Bao et al., 2020) . Next, obtaining clinical tissues of human brain is generally difficult for obvious reasons and it becomes even more complicated to obtain from patients with contagious diseases due to safety concerns (Hanley et al., 2020) . Table 2 summarizes neurotropic viruses studied in the human brain organoids.

To date, only a small number of works have modeled the SARS-CoV-2's neurotropism and neurotoxic effects in human brain organoids (Jacob et al., 2020; Pellegrini et al., 2020a; Pinar Mesci, 2020; Ramani et al., 2020; Yang et al., 2020; Zhang et al., 2020 ) (Eric Song, 2020 Jacob et al., 2020) . Ramani et al. exposed iPSCs-derived brain organoids to SARS-CoV-2. During the early onset of the pandemic in March 2020, the authors could not acquire commercial anti-SARS-CoV-2 antibodies to detect the neurotropic effect of the virus in brain organoids. Instead, they generated convalescent plasma from recovered COVID-19 patients. The convalescent plasma that specifically recognized SARS-CoV-2's spike protein could label the virus in their organoids. Their findings revealed that the virus could directly target a moderate number of cortical neurons expressing pan-neuronal and cortical markers of TUJ-1 and Tau . The authors have tested two age groups of organoids falling forty days apart and identified that the later age groups were much more susceptible to the viral entry. Notably, the tropism of SARS-CoV-2 in their experiments was largely limited to post-mitotic neurons, a property of the virus markedly differing from the Zika virus, which prefers proliferating neural progenitor cells (Gabriel et al., 2017) .

Independent studies by Mesci, Song, Zhang and Yang et al (Pinar Mesci, 2020; Yang et al., 2020; Zhang et al., 2020) have also observed similar neurotropism for SARS-CoV-2 in their brain organoids or iPSCs-derived neurons, i.e., the virus targeting progenitors, neurons, and glial cells except for Pellegrini et al. whose findings state that SARS-CoV-2 cannot target neuronal cell types. Consequently, brain organoids pre-treated with anti-ACE2 antibodies reduced the viral entry, suggesting an influential role for ACE2 in neurons. Although ACE2, a known entry factor for SARS-CoV-2, is expressed in low levels in neurons, the finding showing that the viral entry and its replication has raised a question that there may be additional neuron-specific viral entry factors besides ACE2 and TMPRSS2. Recently, Cantututi-Castelvetri et al. elegantly explained the role of neuropilin-1(NRP1) in mediating efficient binding of the SARS-CoV-2 to ACE2 receptors and thereby viral entry (Cantuti-Castelvetri et al., 2020) . Thus, it is worth testing the expression and the role of NRP1 in brain organoids and various cell types derived from brain organoids.

Besides all of these, Jacob et al. systematically tested the neurotropism of SARS-CoV-2 in various region-specific brain organoids of cortical, hippocampal, hypothalamic, and midbrain regions.

Agreeing with the findings of Ramani et al., Jacob et al. also did not identify a significant increase in viral production between 24 to 72 hpi (Hours post-infection). Taken together, these studies which J o u r n a l P r e -p r o o f employed neural organoids to some extent indicate the viral tropism to human neurons, which is also convincingly substantiated by in the cortical neurons of diseased COVID-19 patients (Song et al., 2020a) . These findings do raise the basic question of how the virus could enter the neurons in brain. One potential possibility is that SARS-CoV-2 has a tropism for nasal epithelial cells and can enter the CNS from the nasal cavity through the olfactory bulb (Sungnak et al., 2020) (Montalvan et al., 2020) .

On the other hand, CNS is protected by the blood-brain-barrier (BBB) and the blood-Cerebrospinal fluid (CSF)-barrier against inflammatory agents and pathogens. Thus, damages in these protective barriers may elicit a passive entry route for SARS-CoV-2. Intriguingly, analyzing hippocampal 

Given the complex neurological defects observed in COVID-19 patients, ranging from headaches, dementia-like syndromes, and encephalitis, SARS-CoV-2 induced effects are rather multifactorial cellular defects. It is difficult to comprehend that 2D or 3D in vitro experimental models of neuronal cultures can mirror these effects. However, brain organoids have surprisingly revealed some useful insights. All of the works that have thus far modeled COVID-19 in brain organoids unambiguously showed that the neurons that harbored virus undergo cell death (Jacob et al., 2020; Pinar Mesci, 2020) Such a Tau missorting has been frequently observed in Tauopathy-related disorders. Extending their analysis, they found that the virus entry is also associated with aberrant Tau phosphorylation.

In particular, they observed that the virus-positive neurons were specifically associated with Tau phosphorylation at the Thr-231 site. Tau Thr-231 phosphorylation is one of the hallmarks of aberrant phosphorylation event inducing neuronal stress and tauopathy-like effects. Intriguingly, it has been documented that Herpes Simplex Virus-1 (HSV-1) targets human neurons and causes aberrant Tau phosphorylation and aggregation, typical of neurodegeneration (Alvarez et al., 2012; Wozniak et al., 2009 ). As discussed above, the observed neurotoxic effects cannot be merely caused by the virus directly but also due to the bystander effects such as inflammatory response induced by infiltrating immune cells. This is due to the fact that SARS-CoV-2 infection has shown great potential in inducing a cytokine storm, which has not only affected the lungs, but also other organs throughout the body.

On performing bulk RNA sequencing, Jacob et al. has identified that besides several factors, SARS-CoV-2-infected choroid plexus organoids express an increased level of inflammatory cytokines, TNF-α, and several interleukins, indicating the triggering of an immune response. The authors have also observed the down regulation of AQP1, AQP4, and SLC22A8, components that are critical to maintain the tight junctions of choroid plexus epithelia. In summary, although organoids are reductionist model systems, they have offered crucial insights into the disease mechanisms of COVID-19, which cannot easily be addressed in biopsy or living human brain samples.

As of present it is essential to note that brain organoids used to model COVID-19 are reductionist models containing mostly cell types for chosen differentiation conditions. These organoids, mainly at the proliferative state, are nearly a perfect model system to study the effect of neurotropic viruses that target neural stem cells. COVID-19 clinical symptoms are rather degenerative-like effects occurring in adult brains harboring mature cell types. Even though the current-state-of-the-J o u r n a l P r e -p r o o f art brain organoids have shown that SARS-CoV-2 has a moderate tropism to cortical neurons, the use of brain organoids in COVID-19 modeling still represents a tip of the iceberg. Thus, generating meaningful neurological COVID-19 modeling requires engineered brain organoids expressing mature cell types of astrocytes, oligodendrocytes, myelinated neurons, and other neuronal cell types expressing ACE2, TMPRSS2, and NRP1. Particular importance should be given to astrocytes and microglia, which may play significant roles in neuroinflammation in response to SARS-CoV-2 infection. It has become clear that cortical neurons will not be functional or viable without glial cell support. Astrocytes are glial cells that play critical roles in CNS homeostasis, synaptogenesis, and, most importantly, mediating immune response by interacting with microglia. Microglia, on the other hand, are the resident immune cells in the brain and functions as a first responder to brain infection. It is known that upon viral infection, microglial cells are quickly activated to transmit pro-inflammatory molecules, reactive oxygen species, and activate astrocytes. Microglia are highly heterogeneous, and their full complement is unexpected in currently existing brain organoids since microglial cells do not originate from neuroectoderm. However, efforts have been made to tailor brain organoids to generate microglia.

For example, omitting SMAD inhibitors, Ormel et al. have shown the presence of reactive microglia in brain organoids, where Iba-1 positive cells overlap with PSD-95, a protein associated with neurons involved in synaptogenesis (Ormel et al., 2018) . Therefore, it would be interesting to test the levels of microglial cells in brain organoids in response to SARS-COV-2 infection. This would help in understanding the immediate effect of immune cells on the neighboring neurons in situ.

Likewise, Ramani et al. have also observed Iba-positive microglial cells and S100β-positive astrocytes in their differentiation protocols. Future experiments generating brain organoids with enriched levels of these cell types will help us dissect if the observed neuronal death is due to bystander effects of pro-inflammatory signals caused by these glial cells upon infection.

Another significant limiting factor in the brain organoids is the lack of vasculatures exhibiting characteristics of Blood-Brain-Barrier (BBB). This is because; one cannot exclude the virus spillage into the CNS via a disrupted BBB. This route is indeed favoring the access of cytokines and infected T cells and monocytes. A readily available example is HIV, another RNA virus possessing neurotropism, which has recently attracted a tremendous public health concern. HIV-1-infected T cells and monocytes cross the BBB, eventually targeting microglia and CNS perivascular macrophages (Bertrand et al., 2019) . It remains unknown whether SARS-CoV-2 follows a similar strategy to target the CNS neurons. Thus, brain organoids with vasculature mimicking the microenvironment of BBB can help to address this question. Vasculatures in brain organoids are unexpected, as endothelial cells do not originate from neuroectoderm. Nonetheless, one does not need to be pessimistic about it. Recent advancements in 3D organoid cultures have generated hybrid organoids of neuroepithelial tissues and vasculatures. Co-culturing of preformed J o u r n a l P r e -p r o o f vasculatures and neurospheres has successfully generated these hybrid organoids. The In-Hyun Park lab took an innovative approach to generate functional vasculatures in the human brain organoids. By overexpressing ETS variant 2, a transcription factor that reprograms cells into endothelial cells, their protocol has generated brain organoids with functional vasculatures (Cakir et al., 2019; Pham et al., 2018) .

In summary, even though the current state-of-the-art brain organoid-based COVID-19 modeling is at the primitive stage, these efforts have been the impetus to advancement the field. It is plausible that brain organoids will reveal unprecedented details when the above-described limitations have been satisfactorily addressed.

The current SARS-CoV-2 pandemic has taken center stage in biomedical science. While the focus to date has been mostly on the respiratory systems, about 30% -40% of the COVID-19 patients developing detrimental CNS defects have surprised the world. These numbers have made us speculate what is primarily causing the patients to succumb to the disease? It is noteworthy that the SARS-CoV outbreak in 2003 stopped suddenly. Thus, studies of human brains from SARS-CoV-infected patients with CNS symptoms are lacking. Besides, the currently available human brain organoids did not exist fifteen years ago. Thus, our knowledge of the neurotoxicity of coronaviruses has been significantly limited.

Thanks to the recently emerged human organoid research, which has already given us critical insights into the neurotropism of SARS-CoV-2. Immediate experiments utilizing the mature state of brain organoids and complex brain organoids harboring vasculatures, choroid plexus, astrocytes assure to dissect the neuropathology of SARS-CoV-2 comprehensively. Hopefully, these advancements will enable brain organoids to replicate the viral progenies reliably. This will allow us to conduct drug-screening studies to identify therapeutic compounds that can stop viral entry, replication, and mitigate the CNS symptoms, which deteriorates the quality of human life.

Identifying anti-SARS-CoV-2 agents is vital, especially given the current status of uncertainties in identifying an effective vaccine.

The authors declare that they have no competing financial interests. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

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