A new study by researchers from Institute of Biology at the University of Campinas-Brazil has provided alarming evidence that the SARS-CoV-2 coronavirus infects astrocytes and to a lesser extent, neurons of the human brain by using the NRP1 (Neuropilin-1) receptors and in the process causes damage to the brain.
The study findings were published in the peer reviewed journal: PNAS
COVID-19 is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the hallmark symptoms of COVID-19 are respiratory in nature and related to pulmonary infection, extrapulmonary effects have been reported in COVID-19 patients (1, 2), including symptoms involving the central nervous system (CNS) (3).
Notably, over 30% of hospitalized COVID-19 patients manifest neurological and even neuropsychiatric symptoms (4, 5), and some present varying degree of encephalitis (6).
There are increasing reports of persistent and prolonged effects after acute COVID-19, a long COVID-19 syndrome characterized by persistent symptoms and/or delayed or long-term complications beyond 4 wk from the onset of symptoms. Some of these persistent symptoms are neuropsychiatric sequelae (3).
One study revealed that more than half of hospitalized patients continued to exhibit neurological symptoms for as long as 3 mo after the acute stage (7). Impaired cognition has also been confirmed in recovered patients after hospitalization (8–11), and neurological impairment is consistent with substantial damage to the nervous system (12).
Previous studies on severe acute respiratory syndrome (SARS) patients reported the presence of the SARS coronavirus in the brain tissue and cerebrospinal fluid of subjects who presented neurological symptoms (13–15). SARS-CoV-2 RNA was also detected in the cerebrospinal fluid of patients with meningitis (16–18). Moreover, alterations in the cerebral cortical region compatible with viral infection (19), a loss of white matter, and axonal injury (20) have all been reported in COVID-19 patients.
In line with the potential neurotropic properties of SARS-CoV-2, recent evidence indicated the presence of viral proteins in brain regions of COVID-19 patients (21, 22) as well as in the brains of K18-ACE2 transgenic mice (22, 23) and Syrian hamsters (24) infected with SARS-CoV-2.
The presence of SARS-CoV-2 in the human brain has been associated with marked astrogliosis, microgliosis, and immune cell accumulation (21). Further indicating the ability of SARS-CoV-2 to infect cells of the CNS, SARS-CoV-2 has also been shown to infect human brain organoid cells in culture (22, 25–27). Recently, SARS-CoV-2 has been found to cross the blood–brain barrier (BBB) in mice (28–30) and in two-dimensional static and three-dimensional microfluidic in vitro models (31, 32), therefore potentially reducing the integrity of the BBB.
Despite the accumulating evidence, an integrated understanding of the cellular and molecular mechanisms involved in SARS-CoV-2 brain infection and the consequent repercussions on brain structure and functionality is lacking. To gain further insight into the neuropathological and neurological consequences of COVID-19 and possible cellular and molecular mechanisms, we performed a broad translational investigation of living patients, postmortem brain samples, and preclinical in vitro and ex vivo models.
Clinical data and brain imaging features of COVID-19 patients were found to be associated with neuropathological and biochemical changes caused by SARS-CoV-2 infection in the CNS. We found that astrocytes are the main sites of viral infection within the CNS.
SARS-CoV-2–infected astrocytes exhibited marked metabolic changes resulting in a reduction of the metabolites used to fuel neurons and build neurotransmitters. Infected astrocytes were also found to secrete unidentified factors that lead to neuronal death. These events could contribute to the neuropathological alterations, neuropsychiatric symptoms, and cognitive impairment observed in COVID-19 patients.
Our study demonstrates structural and functional alterations in the brain tissue of COVID-19 patients, which parallel in vivo findings of cortical atrophy, neuropsychiatric symptoms, and cognitive dysfunctions. A recent longitudinal study with 401 individuals (median age of 62 y, infected between March 2020 and April 2021, scanned pre- and postinfection) reported atrophy in the orbitofrontal and parahippocampal regions and cognitive impairment (determined by Color Trail tests) (48).
The patients we analyzed were infected between March and July 2020 (and therefore, were most likely infected with the original SARS-CoV-2 strain), and we also observed atrophy in the orbitofrontal area and cognitive dysfunction (longer time to perform Color Trail tests and poorer verbal memory task performance). Interestingly, patients with only mild COVID-19 also exhibited cortical atrophy in the superior temporal gyrus, which was previously described in a group of patients with severe SARS-CoV-2 infection (49).
We also observed that higher levels of anxiety symptoms correlated with atrophy of the orbitofrontal cortex, a region previously linked with anxiety disorders (50). Our results suggest that anxiety and depression symptoms are at least partially associated with SARS-CoV-2 infection, a hypothesis supported by a recently discovered association between anxiety and reactive astrogliosis in patients after COVID-19 (51).
This study and other reports showing alterations in brain structure and the manifestation of neurological symptoms in COVID-19 patients (52–54) raise a debate on whether these clinical features are a consequence of peripheral changes or rather, viral invasion of the CNS. Both hypotheses are possible as we detected histopathological alterations associated with SARS-CoV-2 presence in brain tissue collected from 5 deceased patients, while 21 individuals who died of COVID-19 did not show any brain tissue alterations.
However, as the sampling region was small, the possibility remains that other brain regions may exhibit COVID-19–related histopathological alterations. Indeed, the limited number of individuals who exhibited brain alterations associated with CNS SARS-CoV-2 detection and the imprecise and heterogenic nature of postmortem sample collection across studies may explain the discussion regarding the potential correlations between neuroinvasion and COVID-19 symptoms (22, 25–27, 55, 56).
Although some studies failed to detect the virus in the CNS (57), others have found viral particles in the brain (21) localized to the microvasculature and neurons (22), the choroid plexus (58), or meninges (59). In vitro models, such as stem cell–derived neural cells and cerebral organoids, have also demonstrated that SARS-CoV-2 potentially infects brain cells (22, 25–27, 55). H
owever, the magnitude of the CNS infection, its distribution within the brain tissue, and the molecular and cellular bases underlying the phenomenon had not been thoroughly explored. Here, we show that astrocytes are the main site of infection—and possibly, replication—of SARS-CoV-2 in the brains of COVID-19 patients as evidenced by the detection of the viral genome, the SARS-CoV-2 spike protein, and dsRNA in postmortem brain tissue, ex vivo brain slices, and in vitro infected astrocytes. These findings corroborate other studies that showed that astrocytes from primary human cortical tissue and stem cell–derived cortical organoids are susceptible to SARS-CoV-2 infection (22, 55, 60).
Recently, Meinhardt et al. (61) described that SARS-CoV-2 could access the CNS through the neural–mucosal interface in olfactory mucosa, thereby entering the primary respiratory and cardiovascular control centers in the medulla oblongata. Other proposed routes of SARS-CoV-2 neuroinvasion include brain endothelial cells (28–30, 32, 61, 62).
In addition to the inflammatory response produced by SARS-CoV-2 infection, endothelial cell infection could also cause dysfunctions in BBB integrity and facilitate further access of the virus to the brain (28–30, 32, 61, 62). Despite the advances that have already been made, there is still much left to be learned about the routes that SARS-CoV-2 can take to invade the brain and how the virus navigates across different brain regions.
While ACE2 is the best-characterized cellular receptor for SARS-CoV-2 to enter cells via interaction with the viral spike protein, other receptors have also been identified as mediators of infection (63). According to our data and others (21), astrocytes do not express ACE2; rather, they exhibit elevated expression of NRP1, another SARS-CoV-2 spike target that is abundantly expressed in the CNS, particularly in astrocytes (Fig. 5C) (38, 39, 64). When NRP1 is blocked with neutralizing antibodies, SARS-CoV-2 infection in these cells is greatly reduced. These results indicate that SARS-CoV-2 infects in vitro astrocytes via the NRP1 receptor, although this has yet to be confirmed in vivo.
To understand the consequences of SARS-CoV-2 infection in NSC-derived astrocytes, we searched for changes in the proteome in a nonhypothesis-driven fashion. SARS-CoV-2 infection resulted in marked proteomic changes in several biological processes, including those associated with energy metabolism, in line with previous reports on other cell types infected with SARS-CoV-2 (65–67).
Noteworthily, differentially expressed proteins in COVID-19 postmortem brains were enriched for astrocytic proteins more than oligodendrocytes, neurons, or Schwann cells, strengthening our findings that these are the most affected cells by SARS-CoV-2 infection in the human brain. Our proteomic data also evidenced changes in the components of carbon metabolism pathways, particularly glucose metabolism, in both in vitro infected astrocytes and postmortem brain tissue from COVID-19 patients.
Since astrocyte metabolism is key to support neuronal function, changes in astrocyte metabolism could indirectly impact neurons. We found that one of the most critical alterations caused by SARS-CoV-2 infection in astrocytes was a decrease in pyruvate and lactate levels. Lactate exportation is one of the ways that astrocytes support neurons metabolically (63), shuttling this carbon source through the astrocyte–neuron lactate shuttle (ANLS) mechanism.
In the ANLS, neuronal activity also enhances glutamate uptake by astrocytes (68). Lactate itself is essential for the support of neuronal activity and cerebral functions, acting as a neuroprotective agent as well as a key signal to regulate blood flow (69). These metabolic changes, particularly the reductions in lactate and pyruvate associated with decreases in MCT1 and MCT2 expression in SARS-CoV-2–infected astrocytes, support the hypothesis of ANLS disruption.
Moreover, intermediates of glutamine metabolism, such as glutamate and GABA, were also decreased in SARS-CoV-2–infected astrocytes. This said, there were no significant changes in any core intermediates of the TCA cycle. Together with an increased oxygen consumption rate of SARS-CoV-2–infected astrocytes, these results suggest that glycolysis and glutaminolysis are being used to fuel carbons into the TCA cycle to sustain the increased oxidative metabolism of infected astrocytes.
Recently, de Oliveira et al. (24) showed that glutamine levels were also found reduced in mixed glial cells infected with SARS-CoV-2 and that the inhibition of glutaminolysis decreased viral replication and proinflammatory response, further reinforcing that glutamine could be used to fuel the TCA cycle in infected astrocytes.
While astrocyte-derived lactate is required for neuronal metabolism (47, 70), as previously mentioned, glutamine is used in the synthesis of neurotransmitters, such as glutamate and GABA (71). Astrocytes also play a vital role in glutamate-level homeostasis (72, 73) and neurotransmitter recycling, crucial processes for the maintenance of synaptic transmission and neuronal excitability.
At glutamatergic synapses, glutamate uptake by astroglia prevents excitotoxicity (74), whereupon glutamine synthetase converts glutamate to glutamine, which can then be transferred back to neurons, thus closing the glutamate–glutamine cycle (75). At GABAergic synapses, GABA is taken up by astrocytes and first metabolized to glutamate before being converted to glutamine (76).
Given the importance of the metabolic coupling between astrocytes and neurons, alterations in astrocytic glucose and glutamine metabolism are expected to compromise neuronal metabolism and plasticity and synaptic function (77). By 18F-FDG PET analysis, Guedj et al. (78) reported hypometabolism in four different clusters of brain regions in patients suffering from long COVID-19, including the bilateral rectal/orbital gyrus and the olfactory gyrus (78).
As key regulators of CNS metabolism, alterations in astrocyte metabolism contribute to the 18F-FDG PET signal (79–81). Therefore, dysfunctions in astrocyte energy metabolism, like those observed here, could explain, at least partially, the brain hypometabolism in COVID-19 patients (78, 82–85).
In addition to the metabolic changes observed in SARS-CoV-2–infected astrocytes that may lead to neuronal dysfunction, we found that SARS-CoV-2 infection elicits a neurotoxic secretory phenotype in astrocytes that results in increased neuronal death. A similar phenomenon has been observed when astrocytes are activated by inflammatory factors (43, 86, 87).
The alterations in cortical thickness we observed in COVID-19 patients could be explained by this neuronal death, at least partially, as well as by other mechanisms, including reactive astrogliosis and alterations in astrocyte specification and morphogenesis, as previously described in Alzheimer’s disease, autism, and schizophrenia (88–90), as well as epilepsy, autism, and self-injury (91–94).
Matschke et al. (21) reported astrogliosis in 86% of individuals who died following a diagnosis of SARS-CoV-2 infection. In agreement, snRNA-seq data show that the main markers of reactive astrocytes (95) are enriched in samples from the medial frontal cortex of patients with COVID-19 compared with noninfected patients, supporting the hypothesis that reactive astrogliosis is a feature of COVID-19 (SI Appendix, Fig. S11).
Astrocytes are also relevant in the regulation of synapses (and neural networks) and have been linked to the manifestation of depression (96), anxiety symptoms (97), and memory impairment (98), all of which have been observed in our post-COVID infection cohort.
In summary, our findings are consistent with a model in which SARS-CoV-2 is able to reach the CNS of COVID-19 patients, infect astrocytes via NRP1 interaction, and secondarily impair neuronal function and viability.
These changes may contribute to the alterations of brain structure observed here and elsewhere, thereby resulting in the neurocognitive and neuropsychiatric dysfunctions manifested by some patients with COVID-19. Our study comes as a cautionary note that mechanisms of neuroinvasion in fatal COVID-19 could also be operative in mild COVID-19.
However, it is important to note that the study was limited in that neuroimaging and cognitive testing were obtained from a different cohort than fatal COVID-19, in which only a minority of individuals showed evidence of astrocytic invasion. Nonetheless, interventions directed to treat COVID-19 should also envision ways to prevent SARS-CoV-2 invasion of the CNS and/or replication in astrocytes.