key: cord-0884688-erbt3ax6
authors: Vargas, Gabriele; Medeiros Geraldo, Luiz Henrique; Salomão, Natália; Paes, Marciano Vianna; Souza Lima, Flavia Regina; Alcantara Gomes, Flávia Carvalho
title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Glial Cells: insights and perspectives
date: 2020-08-13
journal: Brain Behav Immun Health
DOI: 10.1016/j.bbih.2020.100127
sha: 58d8074999642dcdf611e009f4a6274dba36f5f0
doc_id: 884688
cord_uid: erbt3ax6

In December 2019, a pneumonia outbreak was reported in Wuhan, Hubei province, China. Since then, the World Health Organization declared a public health emergency of international concern due to a growing number of deaths around the globe, as well as unparalleled economic and sociodemographic consequences. The disease called coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel form of human coronavirus. Although coronavirus infections have been associated with neurological manifestations such as febrile seizures, convulsions, change in mental status, and encephalitis, less is known about the impact of SARS-CoV-2 in the brain. Recently, emerging evidence suggests that SARS-CoV-2 is associated with neurological alterations in COVID-19 patients with severe clinical manifestations. The molecular and cellular mechanisms involved in this process, as well as the neurotropic and neuroinvasive properties of SARS-CoV-2, are still poorly understood. Glial cells, such as astrocytes and microglia, play pivotal roles in the brain response to neuroinflammatory insults and neurodegenerative diseases. Further, accumulating evidence has shown that those cells are targets of several neurotropic viruses that severely impact their function. Glial cell dysfunctions have been associated with several neuroinflammatory diseases, suggesting that SARS-CoV-2 likely has a primary effect on these cells in addition to a secondary effect from neuronal damage. Here, we provide an overview of these data and discuss the possible implications of glial cells as targets of SARS-CoV-2. Considering the roles of microglia and astrocytes in brain inflammatory responses, we shed light on glial cells as possible drivers and potential targets of therapeutic strategies against neurological manifestations in patients with COVID-19. The main goal of this review is to highlight the need to consider glial involvement in the progression of COVID-19 and potentially include astrocytes and microglia as mediators of SARS-CoV-2-induced neurological damage.

deaths globally ((OPAS) 2020). The total number of reported COVID-19 infections is probably underestimated since there are mild or asymptomatic cases and considering the impossibility of performing population-wide laboratory diagnoses, especially in lowand middle-income countries.

At first, this virus was shown to cause only an acute lower tract respiratory infection, which could lead to pneumonia; however, multiple organ distress syndrome may occur, which may affect several organs, including the brain, provoking neurological manifestations (Dos Santos et al., 2020; Fotuhi et al., 2020) . Although the mechanisms of brain damage in COVID-19 are poorly understood, other members of the coronavirus family have already been associated with neurological disease , which may give support to the neurotropic behavior of this virus.

In previous epidemics, SARS-CoV was detected in the brain and in the cerebrospinal fluid of patients who presented neurological manifestations (Xu et al., 2005) . Some authors related CoV infections to acute disseminated encephalomyelitis (ADEM) (Algahtani et al., 2016) . Four of twenty three patients with MERS-CoV reported neurological symptoms and were diagnosed with Bickerstaff's encephalitis overlapping with Guillain-Barré syndrome, without any respiratory symptoms (Kim et al., 2017) .

Increasing reports of COVID-19 patient cohorts, although still sparse, have shown a prevalence of neurologic signs and symptoms (Helms et al., 2020; Mao et al., 2020) . The clear and predominant neurological symptom of COVID-19 patients is headache, in up to a third of all patients (Helms et al., 2020; Mao et al., 2020) . Following headache, anosmia and ageusia were quickly described as early symptoms of SARS-CoV-2 infection, although the prevalence of these symptoms in J o u r n a l P r e -p r o o f many factors can contribute to the delirium observed in COVID-19 patients, especially the ones from ICU (intense care unit), such as the prolonged sedation and choice of sedative strategies, immobilization, socio-environmental factors, the most advanced age of the patients presenting more severe forms of the disease and, finally, direct viral CNS infection and induction of neuroinflammation (Kotfis et al., 2020a; Kotfis et al., 2020b) .

As will be discussed in the next sections, considering previous evidence of the neuroinvasive potential of coronaviruses and their ability to infect glial cells, this could be a major mechanism driving the development of delirium in COVID-19 patients.

Microglial and astrocytic activation, either by direct infection or systemic inflammation, can lead to a neurotoxic response which translates into neuronal damage and is clinically manifested as delirium (van Gool et al., 2010; Khan et al., 2020) . Furthermore, previous evidence suggests that glial cells in the aged brain are more prone to generate this neurotoxic response to external pathogens, which would explain the increased prevalence of delirium in the elder patients with SARS-CoV-2 infection.

There are strong pieces of evidence of the neurotropism of coronaviruses, most of them based on the clinical manifestations, although it has not been completely elucidated how SARS-CoV-2 accesses the human CNS (for further review on this topic see (Alam et al., 2020; Dos Santos et al., 2020; Fotuhi et al., 2020) . Two hypotheses to explain SARS-CoV-2 neuroinvasion have been proposed so far (Figure 1 ): (i) via hematogenous access or (ii) via retrograde neuronal routes. In the first one (i), the virus seems to infect endothelial cells (ECs) from the blood-brain-barrier (BBB), epithelial cells of the blood-cerebrospinal fluid barrier (BCSFB) or leukocytes that would promote J o u r n a l P r e -p r o o f supported by findings of viral-like particles in brain capillary endothelium in the frontal lobe tissue of postmortem brain of COVID-19 patients (Chigr et al., 2020 ).

An additional route to be considered is through the lymphatic system. Mouse hepatitis virus (MHV) was reported to disseminate in the brain via cervical and mesenteric lymph nodes in addition to viremia (Barthold and Smith 1992) . The recently described glymphatic system (Plog and Nedergaard 2018) , where glial cells play an important role in the communication between blood and nervous system, suggests that infection of glial cells by SARS-CoV-2 might contribute to virus access to brain parenchyma. It is important to highlight that ACE2 binding affinity of the SARS-CoV-2 spike protein ectodomain is much higher than the SARS-CoV-1 spike protein, about 10-20-fold higher (Wrapp et al., 2020) . These findings may be a possible explanation to the rapid spread of SARS-CoV-2 from human to human (Chan et al., 2020) .

Thus, despite similarities of Sars-CoV-2 and other members of coronavirus family, further studies are required to investigate whether SARS-CoV-2 can access the brain through this pathway. Since it is a fairly new virus, this review is aimed at exposing some information about the neuropathogenesis of coronaviruses, with focus on glial cells, which may help in future studies in the field of SARS-CoV-2 and COVID-19 infections.

Glial cells, such as astrocytes and microglia, have key functions in maintaining brain homeostasis and in the CNS response to insults, whether physical, infectious or these conditions is a general neuroinflammation scenario, characterized by activation of glial cells, production and release of pro-and/or anti-inflammatory cytokines and chemokines, antioxidants, free radicals, and neurotrophic factors. Whether this response collaborates in the development or delay of the disease is still a matter of discussion and is highly dependent on the insult, stage of the disease and brain region.

Emerging evidence from our group and others supports the concept that reactive glial cells, astrocytes and microglia have a duality in their phenotype, neurotoxic or neuroprotective properties, depending on the age, infectious stimuli, and physiological/pathological condition (Moraes et al., 2015; Diniz et al., 2017; Diniz et al., 2019; Matias et al., 2019; do Amaral et al., 2020) . The underlying mechanisms of their activation, cellular interplays and the impact of regional astrocytic and microglial heterogeneity are still a matter of discussion. Given the large number of functions performed by astrocytes and microglia in the neuroinflammatory response, it is to be expected that activation of these cells has a major impact on brain function in virus infection, such as the SARS-CoV-2 infection found in COVID-19 patients.

The rabies virus (RABV) is transmitted to humans through biting, scratching and/or licking of infected animals, which can lead to neurological signs, such as confusion, agitation, hallucinations and delirium (Consales and Bolzan 2007) . Despite neurons being primarily infected, RABV is able to infect microglia and astrocytes, possibly contributing to viral spread and persistence of the virus. In addition, they seem to affect neurons via the release of cytokines or neurotoxins (Ray et al., 1997) . In 2009, the World Health Organization updated the dengue classifications. The categories are dengue without warning signs, dengue with warning signs and severe dengue. In the latter, CNS impairment was included, due to the increased number of cases reporting neurological manifestations. In this context, our group has studied the involvement of glial cells in DENV infection. Microglia cells are speculated to be the major target cells of the virus in the brain. In experimental mice and human fatal cases, they exhibit phenotypic changes suggestive of activation (Ramos et al., 1998) . Although it is not fully understood, some authors defend the idea that microglia have proinflammatory and pathogenic roles leading to neurotoxicity, and consequently, to neurological complications. DENV NS3 antigen was detected in microglial cells of a fetus who died together with the mother, who presented severe complications due to a DENV infection. These cells were responsible for producing RANTES, IFN-γ, MCP-1, and VEGF (Nunes et al., 2019) . In contrast, microglia were already reported as having an antiviral role, inducing CD8-positive cytotoxic T lymphocyte responses (Tsai et al., 2016) . Meanwhile, astrocytes were not infected by DENV in vitro and in vivo, although they were altered in number, size and shape in murine models (Velandia-Romero et al., 2016) , as well as in dengue fatal cases (Salomão et al., 2020) , which could cause alterations in brain homeostasis.

Beyond that, ZIKV became known due to reports of newborns with microcephaly born of infected pregnant women in Brazil. The virus infects fetal microglia and induces J o u r n a l P r e -p r o o f high levels of pro-inflammatory mediators, which could be harmful to the fetus, evolving to congenital Zika syndrome (Rabelo et al., 2018) . In addition, our group previously demonstrated that ZIKV viral antigen was detected in astrocytes, neurons, pyramidal neurons and microglia cells in the cortical region in severe syndrome outcome (Alves-Leon et al., 2019). Fetal brain autopsy showed histopathological alterations related to microglia activation, leading to neuroinflammation and viral dissemination to the brain parenchyma (Mlakar et al., 2016) . In in vitro experiments, microglia are highly susceptible to ZIKV infection. In contrast to what is seen in DENV infection, ZIKV is able to infect astrocytes, leading to morphologic changes (Stefanik et al., 2018) , which may alter the maintenance and permeability of the BBB. Recently, Ledur and collaborators demonstrated that ZIKV infection leads to ROS imbalance, mitochondrial defects and DNA breakage in iPSC-derived human astrocytes. They also detected glial reactivity in mice and in post-mortem brains from infected neonates from the northeast of Brazil, indicating that astrocytes are targets and responsive to ZIKV (Ledur et al., 2020) .

Astrocytes were also reported to be infected by WNV in some fatal cases, inducing neuroinflammatory genes (van Marle et al., 2007) . Together with microglial cells, they were activated, which was evidenced by glial hypertrophy and increased number of the brain. The encephalopathy caused by HIV is characterized by the formation of multinucleated giant cells and microglial nodules, which provoke neurological symptoms in more than 50% of patients not receiving antiretroviral therapy. The neurodegeneration seems to occur mainly due to microglial cell infection and activation, leading to production of cytokines and neurotoxic substances (Alirezaei et al., 2008 ) that stimulate astrocytes, contributing to neuronal injury. Otherwise, microglia also have some neuroprotective roles in the early stages of the disease (Gras et al., 2003) . The astrocytes infected by HIV are particularly found in perivascular regions and are associated with macrophages, contributing to neuropathogenesis (Churchill et al., 2009 ).

Concerning the Coronaviridae family, several lines of evidence show that members of this family are able to infect the brain, including SARS-CoV, the human coronavirus (HCoV), MERS-CoV, and the mouse hepatitis virus (MHV). SARS-CoV-1 has been found in eight brain autopsies of patients by electron microscopy, immunohistochemistry, and real-time reverse transcription (Xu et al., 2005; Netland et al., 2008) .

The human coronavirus (HCoV) OC43 strain seems to be able to infect primary cultures of human astrocytes and microglia. In some astrocytic cell lines, the infection is persistent . In MERS-CoV infections, the cerebrospinal fluid presented high levels of MIF (macrophage migration inhibitory factor) and OPN (osteopontin), two pro-inflammatory cytokines produced by activated microglia (Ichiyama et al., 2009 ).

the study of CNS viral infection. MHV causes different types of diseases, such as hepatitis, enteritis, and encephalomyelitis. In encephalomyelitis, the strain JHMV extensively infects brain cells, including astrocytes and microglia , as discussed in subsequent sections.

Although several lines of evidence support infection of the CNS by coronaviruses, there is less data concerning the impact of these viruses, especially the newly identified SARS-Cov-2, on glial cells. In the next sections, we will present new results on the effects of SARS-CoV-2, mainly on microglia and astrocytes, and we will discuss the role of glial cells as modulators of the COVID-19 neuropathology.

Microglial cells are the resident macrophages from the CNS. They are derived from embryonic erythro-myeloid yolk sac progenitors, which colonize the CNS at early stages of development and are maintained by prolonged cellular longevity and local proliferation rather than peripheral hematopoiesis (Ginhoux et al., 2010) . As microglia are the key components of the innate immune response in the CNS, it is clear that any viral infection of the CNS will drive direct and indirect responses of these cells, which are essential for clearance of viral particles and dying neurons and can be responsible Finally, a key life-threatening clinical presentation of patients with severe COVID-19 is the cytokine storm (Coperchini et al., 2020; Huang et al., 2020; Mehta et al., 2020) . This term was first employed to describe the life-threatening exaggerated and uncontrolled general activation of the immune system observed in severe forms of graftversus-host disease (Tisoncik et al., 2012) and can be observed in several infectious diseases (Tisoncik et al., 2012) . This phenomenon is characterized by the production of high levels of several inflammatory mediators (such as interferons, TNFα, interleukins (particularly IL-1β, IL-6 and IL-10), chemokines and colony-stimulating factors (CSFs) (Tisoncik et al., 2012; Coperchini et al., 2020) , which are increased in patients with severe forms of COVID-19, and is capable of predicting prognosis (Henry et al., 2020; Huang et al., 2020; McGonagle et al., 2020) . In this context, inhibiting IL-6 signaling using neutralizing monoclonal anti-IL-6 antibodies (Tocilizumab) is a strategy currently being tested in several clinical trials with severe COVID-19 patients and showing promising preliminary results .

In this sense, the cytokine storm could also be responsible for some of the 

Astrocytes, one of the largest glial cell populations in the brain, are responsible for controlling several steps of brain development and function in the formation and maturation of synapses (Diniz et al., 2012; Diniz et al., 2014 a; Diniz et al., 2014 b) and maintenance, pruning and remodeling of synapses in development, aging and diseases (Matias et al., 2016; Matias et al., 2019) . Further, they control neurotransmitter release and uptake and production of trophic factors essential for neuronal differentiation and survival. In addition, astrocytes maintain intimate contact along with the vasculature, thus contributing to the formation and function of the BBB (da Silva et al., 2019) and the recently described glymphatic system, through which compounds such as glucose and amino acids are distributed, and the excess of toxic waste products is removed (Plog and Nedergaard 2018) .

Further, astrocytes play a key role in brain injury by triggering a response known as astrocyte reactivity. This is characterized by changes in the profile of astrocytes' gene expression, leading to both morphological and functional changes that lead to the production of several pro-and anti-inflammatory signals. The extension of the astrocytic reaction may vary depending on the nature of the insult, such as stroke, neurodegenerative disorders, tumors, trauma, infection, ischemia and aging; size of the affected area; the intensity of BBB disruption; and the inflammatory response (Sofroniew 2009; Matias et al., 2019) .

Given the large number of functions performed by astrocytes in the healthy and injured brain, it is to be expected that these cells have a major impact on brain damage caused by SARS-CoV-2, either as direct targets of the virus or by controlling the inflammatory response to the virus and BBB rupture.

Recently, a biochemical analysis of the plasma from severe and moderate cases of COVID-19 patients demonstrated enhancement of biomarkers of CNS injury, such as GFAP (glial fibrillary acidic protein) and NfL (neurofilament light chain protein), suggesting astrocyte activation and neuronal injury in these patients (Kanberg et al., 2020) . In the following paragraphs, we will discuss evidence that suggests how astrocytes can contribute to different aspects of SARS-CoV-2 damage: access and spread to the brain, persistence in the organism and inflammatory response.

In order to address SARS-CoV-2 effects on the brain, several studies have taken advantage of experimental models that use other members of the coronavirus family, such as the human respiratory coronavirus (HCV) (Lachance et al., 1998) Although neurotropism has been shown for other coronavirus, so far, infection of glial cells by SARS-CoV-2 still awaits strong evidence, especially in vivo.

As previously discussed, entrance of SARS-CoV-2 into human cells is dependent on ACE2, which triggers the start of the infectious process (Butowt and Bilinska 2020; Galougahi et al., 2020; Ou et al., 2020; Zhou et al., 2020) . Recently, Chen and collaborators, using transcriptome databases, showed that most ACE2 was found in J o u r n a l P r e -p r o o f neuron and non-neuron cells in the human middle temporal gyrus and posterior cingulate cortex . From non-neuron cells, glial cells, mostly astrocytes and oligodendrocytes, were positive for ACE2 in the human brain, while microglia were positive only in the human middle temporal gyrus .

The authors also found high expression of ACE2 in the olfactory bulb and ECs, supporting the hypothesis of access of SARS-CoV-2 into the brain by the olfactory bulb or BBB.

Astrocytes are described as the major CNS cell for coronavirus MHV (Cai et al., 2003; Cai et al., 2006) and human respiratory coronavirus (HCoV) (Pearson and Mims 1985) persistence. Recently, however, by using SARS-CoV-2 infected iPSC-derived human brain organoids, Mesci and collaborators described that astrocytes were infected and showed a 4-fold increase in death, but no viral accumulation was observed. This suggests that although astrocytes might be targets of SARS-CoV-2, these cells may not replicate the virus (Mesci et al., 2020) as suggested for other coronavirus family members (Cai et al., 2003; Cai et al., 2006) . Further, the authors also observed that excitatory synaptogenesis was highly impaired in SARS-CoV-2 infected organoids.

Since astrocytes have key roles in synaptogenesis by secreting several synaptogenic molecules (Diniz et al., 2012; Diniz et al., 2014 a) , it is likely that SARS-CoV-2 may affect astrocyte synaptogenic properties (Figure 1) . However, this remains to be investigated.

Together, these results suggest that, according to cell-type distribution analysis of ACE2 in the human and mouse brain and glial response to murine and human coronavirus, SARS-CoV-2 might be capable of directly infecting several neural cells, J o u r n a l P r e -p r o o f including astrocytes, thus contributing to the neurological manifestations in COVID-19 patients (Figure 1) . Confirmation of glial infection by SARS-CoV-2 in patients, though, awaits further evidence, including in vivo demonstration.

As previously discussed, two pathways are suggested to allow access of SARS-CoV-2 to the CNS: from the nasal cavity or by crossing BBB.

Astrocyte endfeet form the glial surface as part of the BBB. In MHV infections, intracellular distribution of connexin 43 (Cx43) leads to loss of glial-pial gap junction communication and indicates a possible contribution to disruption of the BBB integrity, thus contributing to viral entrance (Basu et al., 2015; Bose et al., 2018) . Furthermore, it is suggested that astrocytes may act as a conduit for the spread of MHV virus between neurons through their synapses . Taken together, those observations suggest that through their role in BBB structure and function, astrocytes may contribute to coronavirus brain access and infection spread (Figure 1) .

Besides astrocyte contributions to SARS-CoV-2 infection and/or persistence, the main involvement of astrocytes in COVID-19 is certainly controlling brain inflammation ( Figure 1) . Although astrocytes can present protective roles by producing antiinflammatory and survival factors, these cells can also acquire a toxic reactive phenotype, thus producing cytokines that exacerbate injuries or presenting impaired/loss of functions, as we will discuss shortly (Diniz et al., 2017) .

By using a sepsis model, our group previously demonstrated that LPS-stimulated microglia induced synaptic elimination, whereas activated astrocytes increased synapse numbers. Both cell types showed increased production of TNF-α and IL-6, and while astrocytes had increased production of TGF-β1, an anti-inflammatory and synaptogenic J o u r n a l P r e -p r o o f cytokine, microglia showed elevated secretion of interleukin-1β (IL-1β), a proinflammatory cytokine (Moraes et al., 2015) .

Further, the profile of cytokines secreted by astrocytes also varies within different contexts of neurodegenerative diseases. We previously demonstrated that astrocyte functions are impaired in an Alzheimer's disease model mainly due to decreased production of the anti-inflammatory and synaptogenic molecule, TGF-β1 (Diniz et al., 2017) . Conversely, increased production of this cytokine by astrocytes is observed in a Parkinson's disease experimental model (Diniz et al., 2019) . These data shed light on the heterogeneity of glial responses to different insults.

In this context, production of cytokines by astrocytes seems to be a determinant of coronavirus neurovirulence and disease behavior. Highly neurovirulent MHV strains induce astrocyte release of pro-inflammatory cytokines, such as interleukin 12 (IL-12), p40, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin-15 (IL-15), and interleukin one beta (IL-1β) (Li et al., 2004) . Further, spinal cord astrocytes also express those major cytokines, TNF-α, IL-6 and IL-1β, as well type 2 nitric oxide synthase (iNOs) and major histocompatibility complex (MHC) class I and II, due to the chronic demyelinating process in virus-infected mice . Also, HCV-OC43 induces upregulation of IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) mRNA expression in an in vitro model with an astrocytic cell line, U-373MG (Edwards et al., 2000) . Together, these findings suggest that coronavirus neurovirulence may depend on the inflammatory profile triggered by astrocyte activation in response to virus infection in the CNS (Figure 1 ).

Recently, by using tissue cultures of microglia and clonal populations of astrocytes, Lavi and Cong found that microglia and type I astrocytes produced proinflammatory cytokines in response to MHV-A59 infection, including IL-α and β, IL-2, IL-15, IL-13, IL-17, IFN family, IL-6, and TNF-α (Lavi and Cong 2020) .

Notably, IL-6-related cytokine storm described in the COVID-19 pathology is highly associated with severity, criticality, viral load, and prognosis of patients (Magro 2020) . Severe inflammation based on release of IL-6 was associated with higher mortality in mice infected with SARS-CoV-1 and also with SARS-CoV-2 in COVID-19 patients (Magro 2020) . Based on this data, the use of a monoclonal antibody against IL-6 (Tocilizumab) has been proposed as a therapeutic alternative to COVID-19.

Taken together, data from MHV models and in vitro assays suggest that astrocytes may be a target and respond to SARS-CoV-2. It is likely that cross talk between astrocytes, microglia and ECs plays a key role in the control of the cytokine microenvironment and brain function in COVID-19. Whether astrocytic activation is beneficial or harmful to COVID-19 pathology is still a matter of investigation. Further, it remains to be determined if SARS-CoV-2 infection impairs astrocyte functions, such as synaptogenesis and neuronal trophic support. Lastly, it is essential to investigate if astrocyte infection is observed in COVID-19 patients and, if so, what are the consequences of the brain inflammatory response elicited by SARS-CoV-2 infection in the long term.

J o u r n a l P r e -p r o o f Astrocytes and microglia play key roles in several events of brain function and in response to injury. As extensively discussed in this review, due to the broad participation of these cells in brain homeostasis and viral infections, it is likely that impaired functions of glial cells may directly or indirectly impact COVID-19 development.

Aging is considered a main risk factor for higher mortality in COVID-19 patients, although the correlation between SARS-CoV-2 and aging is still unclear (Hascup and Hascup 2020) . Age-dependent remodeling is observed in glial cells, which ultimately leads to impairment/loss of functional properties and may contribute in certain cases to the development of neurodegenerative diseases. Some of these alterations include the appearance of A1, a toxic astrocyte phenotype (Clarke et al., 2018) ; upregulation of genes that eliminate synapses and partially resemble reactive astrocytes (Boisvert et al., 2018) ; exacerbation of neuroinflammation; loss of proteostasis and reduction of stress response mechanisms and several other senescent markers (Verkhratsky and Nedergaard 2018; Steardo et al., 2020) .

Similarly, microglial cells also present several morphological and functional disabilities in the aged brain such as reduced phagocytic capacity; increased ROS and pro-inflammatory cytokine production (Koellhoffer et al., 2017) ; loss of dendritic branching and reduced motility (Damani et al., 2011) and senescence-related changes such as increased DNA and mitochondrial damage and telomere shortening (von Bernhardi et al., 2015; Angelova and Brown 2019) .

Further, as consequence of the aging process, inflammaging, a process characterized by an increase of systemic cytokine levels, namely IL-1β, IL-6, and TNFα, and the senescence-associated secretory phenotype (SASP) is started (Akbar and J o u r n a l P r e -p r o o f astrocytes and microglial cells which not only lose their 'normal' neuroprotective role, but they are also more prone to induce neurodegeneration and neurotoxicity (Dilger and Johnson 2008; Norden and Godbout 2013; Lana et al., 2016) . Although this event has not been clearly demonstrated in elderly COVID-19 patients, it is well known that exaggerated release of proinflammatory cytokines causes an amplified neuroinflammation and constitutes the main trigger of systemic symptoms and neurological impairment in COVID-19 patients (Hascup and Hascup 2020; Mao et al., 2020; Montalvan et al., 2020) .

Since glial cells present a molecular signature during aging, it would be of interest to investigate if this signature contributes to the higher vulnerability of the elderly in COVID-19. Interestingly, a database analysis revealed that SARS-CoV-2 proteins interact with human proteins associated with several aging-related processes such as vesicle trafficking, lipid modifications, RNA processing and regulation, ubiquitin ligases, and mitochondrial activity (Gordon et al., 2020) . Several of these pathways are also associated with neurodegenerative diseases such as Alzheimer's and Parkinson's . Thus, this data together with the fact that glial cell dysfunctions are highly associated with aging and neurodegenerative diseases (Diniz et al., 2017; Diniz et al., 2019) highlight the need to consider the long-term consequences of glial activation and neuroinflammation triggered by SARS-CoV-2. Interestingly, recently, Viel and colleagues showed that low-dose of lithium suppresses IL-6 and reduces SASP (senescence-associated secretory phenotype) in senescent human astrocytes, J o u r n a l P r e -p r o o f suggesting that is a potential therapeutic strategy to COVID-19 in elderly patients (Viel et al., 2020) .

Further, several recent studies showed that astrocytes and microglia are very heterogeneous populations of cells both in the healthy brain as well as in response to different insults and upon aging (Soreq et al., 2017; Boisvert et al., 2018; Buosi et al., 2018; Masuda et al., 2020) . The underlying mechanisms of their activation, cellular interplays and the impact of regional glial heterogeneity are still a matter of discussion.

Emerging data have correlated glial diversity to brain region-specific susceptibility to aging and neurodegenerative diseases (Soreq et al., 2017; Angelova and Brown 2019) .

Whether glial heterogeneity and diversity in the CNS contribute to the distinct vulnerability of different brain regions to SARS-CoV-2 remains to be established.

Whether the involvement of glial cells in COVID-19 is directly due to their infection by SARS-CoV-2, thus impairing their regular biological functions, or indirectly, by controlling neuroinflammation, BBB integrity, and virus spread, remains to be investigated. Based on other neurotropic viruses and neurodegenerative diseases where glial involvement is relevant, SARS-CoV-2 likely has direct and indirect effects on glia that play a role in COVID-19. Whether glial activation is beneficial or harmful to the brain in COVID-19 pathology is still a matter of investigation. Thus, there are still many uncertainties and open questions to be addressed, listed in Box 1. Addressing these questions will certainly not only provide a better understanding of glial involvement in SARS-CoV-2 infection but ultimately may contribute to developing glia-based therapeutic strategies for the treatment of COVD-19. (1.1 and 1. 2) or via hematogenic access (2). In both cases, astrocyte involvement is suggested. In the first one, SARS-CoV-2 seems to use the olfactory nerve and then the olfactory bulb to reach the brain by transsynaptic contact between mitral cells and neurons (1.1) and then between neurons and astrocyte's endfeet on the synaptic cleft (1.2). Second, SARS-CoV-2 may infect endothelial cells (ECs) and/or astrocytes from the blood-brain-barrier (BBB), leading to increased permeability and/or rupture of the BBB (2) and further infection of other cells around the neurovascular region (2). Both options lead to viral dissemination through the CNS. SARS-CoV-2 replication by glial cells is unclear, although astrocytes have been suggested as a reservoir of coronavirus and thus contribute to virus spread (3). In response to virus infection, microglial cells trigger T cell and APC activation (4.1) and recruitment/activation of innate immune cells (monocytes, macrophages and APC) (4.2). This scenario leads to release of several cytokines, chemokines and colonystimulating factors (CSFs) by astrocytes and microglia (5), known as a "cytokine storm," which leads to neurotoxicity and synapse loss (6) and consequently may contribute to short-and long-term brain damage in COVID-19 patients. OB, olfactory bub; OR, olfactory receptor; MC, mitral cells; ECs, endothelial cells; ACE2, angiotensin converting enzyme 2.

J o u r n a l P r e -p r o o f 

Folha informativa -COVID-19 (doença causada pelo novo coronavírus)

Aging immunity may exacerbate COVID-19

Neurological Complications of Middle East Respiratory Syndrome Coronavirus: A Report of Two Cases and Review of the Literature

Decreased neuronal autophagy in HIV dementia: a mechanism of indirect neurotoxicity

Zika virus found in brain tissue of a multiple sclerosis patient undergoing an acute disseminated encephalomyelitis-like episode

Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication

Microglia and the aging brain: are senescent microglia the key to neurodegeneration?

Neuroinvasion by human respiratory coronaviruses

Persistent infection of neural cell lines by human coronaviruses

Viremic dissemination of mouse hepatitis virus-JHM following intranasal inoculation of mice

Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

The Aging

Astrocyte Transcriptome from Multiple Regions of the Mouse Brain

Loss of Cx43-Mediated Functional Gap Junction Communication in Meningeal Fibroblasts Following Mouse Hepatitis Virus Infection

Nonneural expression of SARS-CoV-2 entry genes in the olfactory epithelium suggests mechanisms underlying anosmia in COVID-19 patients

The microbiota protects from viral-induced neurologic damage through microglia-intrinsic TLR signaling

Heterogeneity in Synaptogenic Profile of Astrocytes from Different Brain Regions

SARS-CoV-2: Olfaction, Brain Infection, and the Urgent Need for Clinical Samples Allowing Earlier Virus Detection

Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection

Induction of transcription factor Egr-1 gene expression in astrocytoma cells by Murine coronavirus infection

Dengue Virus Infection of Blood-Brain Barrier Cells: Consequences of Severe Disease

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Glial activation involvement in neuronal death by Japanese encephalitis virus infection

The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain

Comment on "The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients

Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia

COVID-19 Outbreak: An Overview

Normal aging induces A1-like astrocyte reactivity

Rabies review: immunopathology, clinical aspects and treatment

The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokinereceptor system

Radial Gliaendothelial Cells' Bidirectional Interactions Control Vascular Maturation and Astrocyte Differentiation: Impact for Blood-brain Barrier Formation

Age-related alterations in the dynamic behavior of microglia

Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system

Brainderived neurotrophic factor levels in late-life depression and comorbid mild cognitive impairment: a longitudinal study

Astrocyte-induced synaptogenesis is mediated by transforming growth factor beta signaling through modulation of D-serine levels in cerebral cortex neurons

Astrocytes and the TGF-beta1 Pathway in the Healthy and Diseased Brain: a Double-Edged Sword

Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling

Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Abeta Oligomers in Alzheimer's Disease Model

Microglial lysophosphatidic acid promotes glioblastoma proliferation and migration via LPA1 receptor

Neuromechanisms of SARS-CoV-2: A Review

Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43

Activation of glial cells by human coronavirus OC43 infection

Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms

SARS-CoV-2 receptor and entry genes are expressed by sustentacular cells in the human olfactory neuroepithelium

Neurobiology of COVID-19

Olfactory Bulb Magnetic Resonance Imaging in SARS-CoV-2-Induced Anosmia: The First Report

T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses

Self-reported olfactory and taste disorders in SARS-CoV-2 patients: a cross-sectional study

Fate mapping analysis reveals that adult microglia derive from primitive macrophages

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Regulated expression of sodium-dependent glutamate transporters and synthetase: a neuroprotective role for activated microglia and macrophages in HIV infection?

Does SARS-CoV-2 infection cause chronic neurological complications

Neurologic Features in Severe SARS-CoV-2 Infection

Hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): a meta-analysis

Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigenpresenting cells

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Differential replication of pathogenic and nonpathogenic strains of West Nile virus within astrocytes

Serum and cerebrospinal fluid levels of cytokines in acute encephalopathy associated with human herpesvirus-6 infection

Vacuolating encephalitis in mice infected by human coronavirus OC43

Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms

Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19

p r o o f of Delirium Duration and Delirium Severity in the ICU

Neurological Complications during Treatment of Middle East Respiratory Syndrome

Neuroinflammation During RNA Viral Infections

Old Maids: Aging and Its Impact on Microglia Function

COVID-19: What do we need to know about ICU delirium during the SARS-CoV-2 pandemic?

COVID-19: ICU delirium management during SARS-CoV-2 pandemic

Involvement of aminopeptidase N (CD13) in infection of human neural cells by human coronavirus 229E

The neuronastrocyte-microglia triad involvement in neuroinflammaging mechanisms in the CA3 hippocampus of memory-impaired aged rats

Interactions of human microglia cells with Japanese encephalitis virus

Type I astrocytes and microglia induce a cytokine response in an encephalitic murine coronavirus infection

Olfactory and gustatory dysfunctions as a clinical presentation of mildto-moderate forms of the coronavirus disease (COVID-19): a multicenter European study

Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes

Infection

Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4

Potential of large "first generation" human-tohuman transmission of 2019-nCoV

Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

SARS-CoV-2: At the Crossroad Between Aging and Neurodegeneration

SARS-CoV-2 and COVID-19: is interleukin-6 (IL-6) the 'culprit lesion' of ARDS onset? What is there besides Tocilizumab? SGP130Fc

Glia expression of MHC during CNS infection by neurotropic coronavirus

Microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system

Neurologic Manifestations of Hospitalized Patients

With Coronavirus Disease

Microglia Heterogeneity in the Single-Cell Era

Functions of flavonoids in the central nervous system: Astrocytes as targets for natural compounds

Astrocyte Heterogeneity: Impact to Brain Aging and Disease

Aging, Male Sex, Obesity, and Metabolic Inflammation Create the Perfect Storm for COVID-19

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage

Activation Syndrome-Like Disease

COVID-19: consider cytokine storm syndromes and immunosuppression

Sofosbuvir protects human brain organoids against SARS-CoV-2

Zika Virus Associated with Microcephaly

Neurological manifestations of COVID-19 and other coronavirus infections: A systematic review

Activated Microglia-Induced Deficits in Excitatory Synapses Through IL-1beta: Implications for Cognitive Impairment in Sepsis

Japanese Encephalitis Virus-induced let-7a/b interacted with the NOTCH-TLR7 pathway in microglia and facilitated neuronal death via caspase activation

Receptor-independent spread of a highly neurotropic murine coronavirus JHMV strain from initially infected microglial cells in mixed neural cultures

Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2

Review: microglia of the aged brain: primed to be activated and resistant to regulation

A Stillborn Multiple Organs' Investigation from a Maternal DENV-4 J o u r n a l P r e -p r o o f Infection: Histopathological and Inflammatory Mediators Characterization

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Differential susceptibility of cultured neural cells to the human coronavirus OC43

The Glymphatic System in Central Nervous System Health and Disease: Past, Present, and Future

Placental Inflammation and Fetal Injury in a Rare Zika Case Associated With Guillain-Barre Syndrome and Abortion

Dengue virus in the brain of a fatal case of hemorrhagic dengue fever

Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes

Psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and meta-analysis with comparison to the COVID-19 pandemic

Fatal Dengue Cases Reveal Brain Injury and Viral Replication in Brain-Resident Cells Associated with the Local Production of Pro-Inflammatory Mediators

Systemic inflammation and the brain: novel roles of genetic, molecular, and environmental cues as drivers of neurodegeneration

Fine Tuning the Cytokine Storm by IFN and IL-10 Following Neurotropic Coronavirus Encephalomyelitis

Molecular dissection of reactive astrogliosis and glial scar formation

North American Brain Expression

Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging

Interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells

Neuroinfection may contribute to pathophysiology and clinical manifestations of COVID-19

Characterisation of Zika virus infection in primary human astrocytes

Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses

Activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus

Spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes

Induction of glial cell MHC antigen expression in neurotropic coronavirus infections. Characterization of the H-2-inducing soluble factor elaborated by infected brain cells

Maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus

Into the eye of the cytokine storm

The role of microglia in the healthy brain

Long-Term Microgliosis Driven by Acute Systemic Inflammation

Microglia retard dengue virus-induced acute viral encephalitis

A clinical perspective of sepsis-associated delirium

Anosmia and Ageusia: Common Findings in COVID-19 Patients

Systemic infection and delirium: when cytokines and acetylcholine collide

West Nile virusinduced neuroinflammation: glial infection and capsid protein-mediated neurovirulence

Endothelial cell infection and endotheliitis in COVID-19

In Vitro Infection with Dengue Virus Induces Changes in the Structure and Function of the Mouse Brain Endothelium

Physiology of Astroglia

Microdose lithium reduces cellular senescence in human astrocytes -a potential pharmacotherapy for COVID-19?

Microglial cell dysregulation in brain aging and neurodegeneration

SARS-CoV-2 infects T lymphocytes through its spike protein-mediated membrane fusion

Coronavirus pathogenesis

Microglia are required for protection against lethal coronavirus encephalitis in mice

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Nervous system involvement after infection with COVID-19 and other coronaviruses

Detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis

Effective treatment of severe COVID-19 patients with tocilizumab

Clinical characteristics and outcomes of patients with severe covid-19 with diabetes

Microglial Phagocytosis of Neurons: Diminishing Neuronal Loss in Traumatic, Infectious, Inflammatory, and Autoimmune CNS Disorders

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Enhanced viral clearance and reduced leukocyte infiltration in experimental herpes encephalitis after intranasal infection of CXCR3-deficient mice