Current opinion in neurological manifestations of SARS-CoV-2 infection.

Neurological symptoms occur in approximately one-third of hospitalized patients with coronavirus disease 2019 (COVID-19). Among these symptoms, hypoxic encephalopathy develops in one-fifth of severe cases, while ischemic strokes due to thrombotic complications are common in one-third of COVID-19 intensive care patients. Brain involvement of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is eventuated by several routes, including hematogenous spread, transsynaptic entry through infected neurons, olfactory nerve, ocular epithelium, vascular endothelium, and impaired blood-brain barrier. Besides the high angiotensin-converting enzyme-2 (ACE2) binding affinity, and FURIN preactivation, SARS-CoV-2 maintains efficient neuronal entry while evading immune surveillance by using basigin and neuropilin-1 receptors. However, the neurological manifestations and their pathogenic mechanisms are still debated in COVID-19 patients.

Introduction

Although severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) mainly targets the epithelial cells of the respiratory tract, resulting in diffuse alveolar damage, among the other tissues brain cells are also potential target of viral attack [1]. In this respect, neurological abnormalities have been described in approximately one-third of patients who required hospitalization for coronavirus disease 2019 (COVID-19) [2]. Neurological symptoms appear largely with nonspecific clinical manifestation. In SARS-CoV-2 infection, these seem to be dependent on a successive series of events such as excessive neuro-inflammation due to cytokine storm, hypoxia, blood pressure imbalance because of the angiotensin II converting enzyme receptor-2 (ACE2) involvement, and severe ischemic stroke due to diffuse intravascular coagulation [2,3]. According to epidemiological studies, acute symptomatic seizures are observed in more than one-fourth of COVID-19 patients. Hypoxic encephalopathy and ischemic strokes due to thrombotic complications indicate the frequent brain involvement in COVID-19 intensive care unit patients, by one-fifth and one-third, respectively [4, 5∗, 6∗∗]. In this context, the most serious neurological signs seen in COVID-19 patients occur with widespread microthrombi and infarction patches in the brain. These histopathological findings are explicated with the increase in the susceptibility to cerebrovascular events due to hypercoagulability [7]. Recent evidences strongly suggest that clinical findings regarding the mechanisms of neurological symptoms caused by SARS-CoV-2 invasion of the central nervous system (CNS) have not yet been clarified and systematically classified. In COVID-19 patients, the role of ACE2 for the transsynaptic spread of the virus in the CNS, the effect of the hyperimmune response on the spread across the blood–brain barrier (BBB) and the persistence of SARS-CoV-2 in the brain are still debated [8].

Potential routes of brain entry

Various assumptions regarding the entry of SARS-CoV-2 into the CNS are widely discussed in recent studies. In SARS-CoV-2-infected patients, it is proposed that neuro-invasion is eventuated by several routes; hematogenous transportation of infected immune cells via the circulatory system of the brain tissue, transsynaptic entry through the infected neurons or olfactory nerve, entry from ocular epithelium, and the virus invasion from the impaired BBB [9,10] (Figure 1 ). Although ACE2 and transmembrane protease serine 2 (TMPRSS2) have been localized to epithelial cells in the nasal mucosa, whether the virus entry is limited to the olfactory epithelium or attacks the olfactory neurons is controversial [9,11]. Despite the olfactory neuron seems to be one of the major routes for the virus spreads, dissemination of the SARS-CoV-2 via hematological pathway has been reported in various studies with widely ranging frequencies between 1% and 41% [12,13]. Indeed, there have been few comprehensive studies, which identify the neuro-invasive potential of the SARS-CoV-2. In postmortem studies of COVID-19 patients, virus detection in frontal lobe neurons, presence of viral particles in brain capillary endothelial cells despite negative polymerase chain reaction testing of the cerebrospinal fluid and increase in plasma biomarkers due to astrocytic or neuronal injury have been attributed to the neural invasion. Thus, rising in glial fibrillary acidic protein (GFAp) level as a marker of astrocytic activation/injury and increasing neurofilament light chain protein (NfL) concentration as a marker of intra-axonal neuronal injury due to brain damage indicate the hematogenous spread of virus [14,15].

Figure 1

Potential entry routes of SARS-CoV-2 to the central nervous system (CNS) and the mechanisms of neuronal damage. Taken through the olfactory pathway, virus would have access to the CNS using transneuronal/synaptic routes and reaches to brain respiratory center. In the hematogenous entry route, endothelial cells may become infected, microthrombi develop due to endothelial dysfunction. CNS infection arises with the SARS-CoV-2 entrance to the brain following blood–brain barrier disruption by cytokine storm. Astrocytes incorporate the viruses either via direct contact with infected endothelial cells, or ACE2 receptors. In response to virus infection, microglial cells trigger T cell, antigen-presenting cell activation and induce synapse loss. Following neuro-invasion and replication of SARS-CoV-2, impairment of ACE2 activity due to exploitation by SARS-CoV-2, activation of AT1R by Ang II, and glutamate mediated inhibition of ACE2 activity contribute to the development of neurotoxicity and neuroinflammation. Excessive extracellular glutamate accumulation in SARS-CoV-2 infection triggers oxidative stress and neuroinflammation via NMDA receptors, while SARS-CoV-2 infection targets glutathione (GSH) biosynthesis and makes the patients more vulnerable to the detrimental effects of virus by decreasing GSH levels (Abbreviations: ACE2: angiotensin II–converting enzyme receptor-2; AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; AngII: angiotensin II; AT1R: Ang II receptor type 1; BBB: blood–brain barrier; BSG/CD147: basigin; CICR: calcium-induced calcium release; CR: cytokine receptor; GSH: reduced glutathione; mt: mitochondria; NMDAR: N-methyl-d-aspartate receptor; nNOS: neuronal nitric oxide synthase; NOX: nicotinamide adenine dinucleotide phosphate oxidase; NRP1: neuropilin 1; ROS: reactive oxygen species; SARS-CoV-2: acute respiratory syndrome-coronavirus-2).

Furthermore, interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF), and IL-17, the cytokines, which are elevated during cytokine storm associated with SARS-CoV-2 infection, lead to the disruption of the BBB and facilitate the entry of the virus to brain. Whereupon SARS-CoV-2 can infect neurons and glial cells through ACE2 receptors and launches a neuroinflammatory response with reactive astrogliosis, and microglial activation [16, 17∗∗, 18]. Nevertheless, it is claimed that certain neurological complications of COVID-19 are due to increased penetration of proinflammatory cytokines from impaired BBB rather than viral entry. Cytokine release–driven neuroinflammatory response that can activate microglial cells in the CNS cause to the release of astroglial marker, S100B protein, which reflects the increase in the BBB permeability [19,20]. As in other parts of the body, the ACE2 receptor is expressed in the brain by endothelial, neuronal, and glial cells. After the spreading of virus to CNS, it contacts with ACE2 on neurons, glia, and vascular endothelium [21,22]. However, it has been suggested that receptors such as neuropilin-1 (NRP1) play a role in viral entry in neurons lacking ACE2 expression [23].

Functions of receptors in brain invasion of SARS-CoV-2

As a rule, SARS-CoV-2 uses ACE2 as the principal binding receptor and is required for proteolytic processing of the spike (S) protein by TMPRSS2 for cell entry [24]. Although data related to ACE2, TMPRSS2, and furin distributions in the olfactory pathway are insufficient, these cells seem to express ACE2, but not TMPRSS2 or furin. But deterioration of the sense of smelling in COVID-19 patients is simultaneous with the emergence of neurological symptoms [25,26]. Actually, the ACE2 is intensely expressed in the pons and medulla oblongata of the human brainstem, which consists of the medullary respiratory centers of the brain. This finding partly elucidates the mechanism of severe respiratory distress in many COVID-19 patients [27].

Considering the above data, the expression of ACE2 receptor in the brain indicates that SARS-CoV-2 has a neuro-invasive ability [28]. Following SARS-CoV-2 entry to the CNS, ACE2 receptors mediate the development of neurotoxicity, neuroinflammation, and neurodegeneration via ensuring viral entrance and replication [29]. SARS-CoV-2 S protein binds to its receptor ACE2 via its receptor-binding domain (RBD) and is proteolytically activated by human proteases. SARS-CoV-2 is preactivated by proprotein convertase FURIN, diminishing its reliance on target cell proteases for entry. But a certain amount of TMPRSS2 is still needed. The high ACE2 binding affinity of the RBD, FURIN preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient neuronal entry while evading immune surveillance [30]. This means that TMPRSS2 is simultaneously activated to allow the coronavirus S protein to enter the cell [24]. It should be added to the activation of TMPRSS2, that the FURIN/PCSK3 cleavage of the S protein facilitates the interaction of SARS-CoV-2 with the ACE2 receptor and viral invasion [31]. Another route is S protein, which also binds to alternative or the additive molecule basigin (CD147/BSG) (Figure 1). This molecule is a novel receptor glycoprotein of the immunoglobulin super family and serves as the mediating factor of the viral invasion [32]. SARS-CoV-2 can also use basigin [33] and NRP1 as well as ACE2 during entry into neurons [23]. Proteases such as TMPRSS11  A/B, cathepsin B and L and FURIN allow viral cell entry and replication via NRP1 docking receptors [30].

Unexpectedly, ACE2 expression significantly declines in the brain of individuals with Alzheimer's disease (AD). This deficit is dependent on the increased amyloid-beta (Aβ) peptide load and tau pathology and hyperactivity of the classical axis of the renin–angiotensin system [34,35]. Apart from AD, the presence of ACE2 has not been demonstrated in many neurons [36], despite increasing reports of neurological symptoms being common in COVID-19 patients [2]. However, apolipoprotein E (ApoE) is a potential link between AD and COVID-19. ApoE, as a Aβ chaperone, confers a 14-fold increase in susceptibility to AD, while constitutes a risk factor for severe SARS-CoV-2 infection and provides a twofold increase in neural entry of virus [37,38]. Paradoxically, having a decrease in ACE2 function in aged patients, coupled with the age-associated reduction in mitochondrial functions make the host more vulnerable to SARS-CoV-2 infection. Considering the more severe course of COVID-19 in elderly patient populations, these observations suggest that a new explanation is needed for the viral entry mechanism [39,40]. These findings support the claim that ACE2 is not the sole gateway for entry of SARS-CoV-2. In this context, it is suggested that the SARS-CoV-2 enters the neuron by binding with its S protein to the NRP1 receptor. Thus, NRP1, FURIN, and TMPRSS11A levels are elevated in SARS-CoV-2-infected cells of these patients. As mentioned above, the olfactory epithelial cells show high expression of NRP1 in COVID-19 patients [23,41]. In addition, vascular endothelial growth factor-A (VEGF-A) is a physiological ligand for the b1b2 pocket in NRP1 [42]. After brain invasion, primary effect of SARS-CoV-2 emerges on glial cells. Secondary effect results in neuronal damage [43].

In the brain, through the conversion of angiotensin II (Ang II) into angiotensin-(1–7) (Ang-(1–7)), ACE2 decreases oxidative stress and alleviates neuronal apoptosis, besides the maintenance of BBB function [44,45] (Figure 1). Contrarily, impairment of ACE2 activity due to exploitation by SARS-CoV-2 contributes to the development of neuroinflammation and apoptosis [46]. By activating Ang II receptor type 1 (AT1R), Ang II stimulates both internalization and ectodomain shedding of ACE2. Thus, ACE2 expression in addition to its enzymatic activity are inhibited [47,48]. Furthermore, cytotoxic levels of glutamate can significantly impair ACE2 activity in cortical neurons and induces excitotoxic damage in the nerve cells. Thereby following endothelial dysfunction, ischemic stroke, and neuronal injury develop [49].

The neurotoxicity of glutamate is launched mainly by an ample amount of Ca2+ influx emerging from overstimulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR1 [50]. The increase in the intracellular [Ca2+] can mainly lead to mitochondrial dysfunction and reactive oxygen species (ROS) generation [51,52]. Because SARS-CoV-2 infection targets glutathione (GSH) biosynthesis, decreased GSH, and selenoprotein levels make the patients more vulnerable to the detrimental effects of virus-induced oxidative stress and proteolysis [53]. The excitotoxicity of excessive glutamate accumulation participates in the progression of inflammatory neurodegeneration via oxidative stress and/or inducible nitric oxide synthase (iNOS)-mediated mechanisms [54,55]. N-methyl-d-aspartate (NMDA) receptor activation due to excess glutamate causes neuronal damage by increasing oxidative stress and inflammation, unlike alpha7 nicotinic acetylcholine receptor (α7 nAChR) [55,56] (Figure 1). Against excessive extracellular glutamate accumulation triggered by diverse inflammatory and oxidative processes, memantine protects cells from glutamate excitotoxicity by blocking the extrasynaptic NMDA receptors [57]. Because memantine is an antagonist of NMDA receptors, it diminishes the SARS-CoV-2-related oxidative stress and inflammation in neurons [58].

Neurological manifestations of COVID-19

In the clinical studies of the hospitalized COVID-19 patients groups consisting of 214 and 841 cases, neurologic manifestations have been observed in 36.4% and 57.4% of the cases, respectively [2,59].

It is still debated whether or not ACE2 is the main route of entry of SARS-CoV-2 into neuronal cells and what strategies might block viral infection, and whether there are humoral antibody responses against SARS-CoV-2 in the CNS of infected patients. Since in some patients with COVID-19 and neurological symptoms, there is robust antibody response to the virus within the CSF, in patients with mild COVID-19, neurological manifestation is frequently emerged as nonspecific signs like dizziness by 7% and headache by 14% [60]. It is thought that the loss of taste and smell is based on the spreading of the virus to the olfactory neurons, however, due to the lack of convincing proof, it is claimed that disruption of sensory-neural mechanisms may depend on the SARS-CoV-2 encephalitis [2,60]. Nevertheless, the prevalence of hypogeusia and hyposmia was 5.6% and 5.1%, respectively [2]. In fact, it is thought that increase in the olfactory and gustatory dysfunctions are directly correlated with the increase in levels of IL-6 in COVID-19 cases [61]. Although the recovery times in most cases are accompanied by simultaneous swab negativization, and normalization of IL-6 levels, in one-fourth of cases taste and smell dysfunctions may persist a longer period after the recovery [61,62]. Actually, these manifestations have been linked to the infection of cell receptors with the virus rather than possible neurological or ischemic damage [61]. Actually, COVID-19 cases involving the CNS resembles traditional cases of acute disseminated encephalomyelitis [63] and acute necrotizing hemorrhagic encephalopathy [64]. Recently, it has been reported that Guillain–Barré syndrome, which occurs with immune attack on peripheral nerves, has been observed in COVID-19 cases [65]. ACE2 is expressed on both surface membrane and in the cytoplasm of some neurons, astrocytes, and oligodendrocytes. Expression of ACE2 is also densely localized in the substantia nigra, ventricles, middle temporal gyrus, posterior cingulate cortex, and olfactory bulb [66]. COVID-19 patients have increased Ang II. The overexpression of ACE2 and the disturbance of balance due to increased Ang II enhances the mortality of COVID-19 patients [3]. Acute ischemic stroke (AIS) has been described in approximately 1–3% of patients with COVID-19 [2,67,68]. Intracranial hemorrhage is seen in about 0.5% of the patients with COVID-19 in large population studies [2,59]. Increase in procoagulant factors in COVID-19 patients, such as serum levels of fibrinogen, platelet, IL-6 and D-dimer, contribute thromboembolic complications and higher rate of mortality and morbidity [69]. ACE2 exploited by SARS-CoV-2, and high Ang II cause endothelial dysfunction in the cerebral arteries, by increasing blood pressure (approximately one-third of all patients) and creating the risk of cerebral hemorrhage and hemorrhagic stroke [70,71]. In these cases, downregulation of the ACE2 receptors and elevated Ang II levels are proportional with viral load and mortality rate [3,72]. AIS rate associated with an inflammatory and hypercoagulable state is observed up to 5% of patients with severe COVID-19. In these patients, the risk of death due to AIS reaches up to 38% [73]. Coagulopathy underlies the occurrence of stroke. Cytokine storm-mediated by SARS-CoV-2 can produce the hypercoagulable state, causes vascular wall damage, and precipitates microthrombosis [74]. Furthermore, the overactivation of the complement cascade, and its interactions with cytokines plays important role in cerebrovascular thrombosis. However, precise data on this subject could not be yet acquired [75,76], ACE2 receptor has an important role in developing neurologic manifestations of SARS-CoV-2 infection. Cerebral hemorrhage and stroke aggravate COVID-19 outcomes [77]. Protecting patients from AIS, which is one of the most dangerous complications of COVID-19 and causes the lethality of more than a third of severe cases, requires a complex treatment program. These include immunomodulatory drugs, anti-inflammatory drugs, and prophylactic antibodies, antiviral drugs and vaccines; however, the most suitable treatment combination is still being under investigation.

Conclusion

The large number of sensitive transmembrane receptors make most of the human cells highly susceptible to the risk of SARS-CoV-2 infection and viral attack. The serious danger of SARS-CoV-2 infection is not only due to its extremely contagious behavior but also due to its attack to many vital and critical human cell and tissue types simultaneously and is deadly. Because the neurological symptoms and their mechanisms could not be comprehensively clarified, in COVID-19 patients, the spread and persistence of the virus in the brain cells are still under debate.

Credit author statement

Ayse Basak Engin: Writing – original draft preparation, Evren Doruk Engin: Reviewing and editing Atilla Engin: Figure preparation, reviewing and editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.