ACE-2, TMPRSS2, and Neuropilin-1 Receptor Expression on Human Brain Astrocytes and Pericytes and SARS-CoV-2 Infection Kinetics.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and "in vitro" viral kinetics in astrocytes and pericytes may improve our understanding of viral infection "in vivo". In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.

SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

Loss of neuron network coherence induced by virus-infected astrocytes: a model study.

Coherent activations of brain neuron networks underlie many physiological functions associated with various behavioral states. These synchronous fluctuations in the electrical activity of the brain are also referred to as brain rhythms. At the cellular level, rhythmicity can be induced by various mechanisms of intrinsic oscillations in neurons or the network circulation of excitation between synaptically coupled neurons. One specific mechanism concerns the activity of brain astrocytes that accompany neurons and can coherently modulate synaptic contacts of neighboring neurons, synchronizing their activity. Recent studies have shown that coronavirus infection (Covid-19), which enters the central nervous system and infects astrocytes, can cause various metabolic disorders. Specifically, Covid-19 can depress the synthesis of astrocytic glutamate and gamma-aminobutyric acid. It is also known that in the post-Covid state, patients may suffer from symptoms of anxiety and impaired cognitive functions. We propose a mathematical model of a spiking neuron network accompanied by astrocytes capable of generating quasi-synchronous rhythmic bursting discharges. The model predicts that if the release of glutamate is depressed, normal burst rhythmicity will suffer dramatically. Interestingly, in some cases, the failure of network coherence may be intermittent, with intervals of normal rhythmicity, or the synchronization can disappear.

Astrocytes in the pathophysiology of neuroinfection.

Key homeostasis providing cells in the central nervous system (CNS) are astrocytes, which belong to the class of cells known as atroglia, a highly heterogeneous type of neuroglia and a prominent element of the brain defence. Diseases evolve due to altered homeostatic state, associated with pathology-induced astroglia remodelling represented by reactive astrocytes, astroglial atrophy and astrodegeneration. These features are hallmarks of most infectious insults, mediated by bacteria, protozoa and viruses; they are also prominent in the systemic infection. The COVID-19 pandemic revived the focus into neurotropic viruses such as SARS-CoV2 (Coronaviridae) but also the Flaviviridae viruses including tick-borne encephalitis (TBEV) and Zika virus (ZIKV) causing the epidemic in South America prior to COVID-19. Astrocytes provide a key response to neurotropic infections in the CNS. Astrocytes form a parenchymal part of the blood-brain barrier, the site of virus entry into the CNS. Astrocytes exhibit aerobic glycolysis, a form of metabolism characteristic of highly morphologically plastic cells, like cancer cells, hence a suitable milieu for multiplication of infectious agent, including viral particles. However, why the protection afforded by astrocytes fails in some circumstances is an open question to be studied in the future.

Neuroinflammation in neurodegeneration via microbial infections.

Recent epidemiological studies show a noticeable correlation between chronic microbial infections and neurological disorders. However, the underlying mechanisms are still not clear due to the biological complexity of multicellular and multiorgan interactions upon microbial infections. In this review, we show the infection leading to neurodegeneration mediated by multiorgan interconnections and neuroinflammation. Firstly, we highlight three inter-organ communications as possible routes from infection sites to the brain: nose-brain axis, lung-brain axis, and gut-brain axis. Next, we described the biological crosstalk between microglia and astrocytes upon pathogenic infection. Finally, our study indicates how neuroinflammation is a critical player in pathogen-mediated neurodegeneration. Taken together, we envision that antibiotics targeting neuro-pathogens could be a potential therapeutic strategy for neurodegeneration.

Morphological, cellular, and molecular basis of brain infection in COVID-19 patients.

Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. We show the spectrum of cerebral impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, ranging from long-term alterations in mildly infected individuals (orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms) to severe acute damage confirmed in brain tissue samples extracted from the orbitofrontal region (via endonasal transethmoidal access) from individuals who died of COVID-19. In an independent cohort of 26 individuals who died of COVID-19, we used histopathological signs of brain damage as a guide for possible SARS-CoV-2 brain infection and found that among the 5 individuals who exhibited those signs, all of them had genetic material of the virus in the brain. Brain tissue samples from these five patients also exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a noncanonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that reduces neuronal viability. Our data support the model in which SARS-CoV-2 reaches the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients.

SARS-CoV-2 infection impacts carbon metabolism and depends on glutamine for replication in Syrian hamster astrocytes.

COVID-19 causes more than million deaths worldwide. Although much is understood about the immunopathogenesis of the lung disease, a lot remains to be known on the neurological impact of COVID-19. Here, we evaluated immunometabolic changes using astrocytes in vitro and dissected brain areas of SARS-CoV-2 infected Syrian hamsters. We show that SARS-CoV-2 alters proteins of carbon metabolism, glycolysis, and synaptic transmission, many of which are altered in neurological diseases. Real-time respirometry evidenced hyperactivation of glycolysis, further confirmed by metabolomics, with intense consumption of glucose, pyruvate, glutamine, and alpha ketoglutarate. Consistent with glutamine reduction, the blockade of glutaminolysis impaired viral replication and inflammatory response in vitro. SARS-CoV-2 was detected in vivo in hippocampus, cortex, and olfactory bulb of intranasally infected animals. Our data evidence an imbalance in important metabolic molecules and neurotransmitters in infected astrocytes. We suggest this may correlate with the neurological impairment observed during COVID-19, as memory loss, confusion, and cognitive impairment.

In vitro neurotoxicity evaluation of biocidal disinfectants in a human neuron-astrocyte co-culture model.

Biocidal disinfectants (BDs) that kill microorganisms or pathogens are widely used in hospitals and other healthcare fields. Recently, the use of BDs has rapidly increased as personal hygiene has become more apparent owing to the pandemic, namely the coronavirus outbreak. Despite frequent exposure to BDs, toxicity data of their potential neurotoxicity (NT) are lacking. In this study, a human-derived SH-SY5Y/astrocyte was used as a co-culture model to evaluate the chemical effects of BDs. Automated high-content screening was used to evaluate the potential NT of BDs through neurite growth analysis. A set of 12 BD substances classified from previous reports were tested. Our study confirms the potential NT of benzalkonium chloride (BKC) and provides the first evidence of the potential NT of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB). BKC and PHMB showed significant NT at concentrations without cytotoxicity. This test system for analyzing the potential NT of BDs may be useful in early screening studies for NT prior to starting in vivo studies.

Enhancing GAT-3 in thalamic astrocytes promotes resilience to brain injury in rodents.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Potential role of astrocyte angiotensin converting enzyme 2 in the neural transmission of COVID-19 and a neuroinflammatory state induced by smoking and vaping.

BACKGROUND: Knowledge of the entry receptors responsible for SARS-CoV-2 is key to understand the neural transmission and pathogenesis of COVID-19 characterized by a neuroinflammatory scenario. Understanding the brain distribution of angiotensin converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, remains mixed. Smoking has been shown as a risk factor for COVID-19 severity and it is not clear how smoking exacerbates the neural pathogenesis in smokers. METHODS: Immunohistochemistry, real-time PCR and western blot assays were used to systemically examine the spatial-, cell type- and isoform-specific expression of ACE2 in mouse brain and primary cultured brain cells. Experimental smoking exposure was conducted to evaluate the effect of smoking on brain expression. RESULTS: We observed ubiquitous expression of ACE2 but uneven brain distribution, with high expression in the cerebral microvasculature, medulla oblongata, hypothalamus, subventricular zones, and meninges around medulla oblongata and hypothalamus. Co-staining with cell type-specific markers demonstrates ACE2 is primarily expressed in astrocytes around the microvasculature, medulla oblongata, hypothalamus, ventricular and subventricular zones of cerebral ventricles, and subependymal zones in rhinoceles and rostral migratory streams, radial glial cells in the lateral ventricular zones, tanycytes in the third ventricle, epithelial cells and stroma in the cerebral choroid plexus, as well as cerebral pericytes, but rarely detected in neurons and cerebral endothelial cells. ACE2 expression in astrocytes is further confirmed in primary cultured cells. Furthermore, isoform-specific analysis shows astrocyte ACE2 has the peptidase domain responsible for SARS-CoV-2 entry, indicating astrocytes are indeed vulnerable to SARS-CoV-2 infection. Finally, our data show experimental tobacco smoking and electronic nicotine vaping exposure increase proinflammatory and/or immunomodulatory cytokine IL-1a, IL-6 and IL-5 without significantly affecting ACE2 expression in the brain, suggesting smoking may pre-condition a neuroinflammatory state in the brain. CONCLUSIONS: The present study demonstrates a spatial- and cell type-specific expression of ACE2 in the brain, which might help to understand the acute and lasting post-infection neuropsychological manifestations in COVID-19 patients. Our data highlights a potential role of astrocyte ACE2 in the neural transmission and pathogenesis of COVID-19. This also suggests a pre-conditioned neuroinflammatory and immunocompromised scenario might attribute to exacerbated COVID-19 severity in the smokers.

Central Nervous System Pericytes Contribute to Health and Disease.

Successful neuroprotection is only possible with contemporary microvascular protection. The prevention of disease-induced vascular modifications that accelerate brain damage remains largely elusive. An improved understanding of pericyte (PC) signalling could provide important insight into the function of the neurovascular unit (NVU), and into the injury-provoked responses that modify cell-cell interactions and crosstalk. Due to sharing the same basement membrane with endothelial cells, PCs have a crucial role in the control of endothelial, astrocyte, and oligodendrocyte precursor functions and hence blood-brain barrier stability. Both cerebrovascular and neurodegenerative diseases impair oxygen delivery and functionally impair the NVU. In this review, the role of PCs in central nervous system health and disease is discussed, considering their origin, multipotency, functions and also dysfunction, focusing on new possible avenues to modulate neuroprotection. Dysfunctional PC signalling could also be considered as a potential biomarker of NVU pathology, allowing us to individualize therapeutic interventions, monitor responses, or predict outcomes.

Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes.

Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.

Molecular Mechanisms of Proteins - Targets for SARS-CoV-2 (Review).

The rapidly accumulating information about the new coronavirus infection and the ambiguous results obtained by various authors necessitate further research aiming at prevention and treatment of this disease. At the moment, there is convincing evidence that the pathogen affects not only the respiratory but also the central nervous system (CNS). The aim of the study is to provide an insight into the molecular mechanisms underlying the damage to the CNS caused by the new coronavirus SARS-CoV-2. Results: By analyzing the literature, we provide evidence that the brain is targeted by this virus. SARS-CoV-2 enters the body with the help of the target proteins: angiotensin-converting enzyme 2 (ACE2) and associated serine protease TMPRSS2 of the nasal epithelium. Brain damage develops before the onset of pulmonary symptoms. The virus spreads through the brain tissue into the piriform cortex, basal ganglia, midbrain, and hypothalamus. Later, the substantia nigra of the midbrain, amygdala, hippocampus, and cerebellum become affected. Massive death of neurons, astrogliosis and activation of microglia develop at the next stage of the disease. By day 4, an excessive production of proinflammatory cytokines in the brain, local neuroinflammation, breakdown of the blood-brain barrier, and impaired neuroplasticity are detected. These changes imply the involvement of a vascular component driven by excessive activity of matrix metalloproteinases, mediated by CD147. The main players in the pathogenesis of COVID-19 in the brain are products of angiotensin II (AT II) metabolism, largely angiotensin 1-7 (AT 1-7) and angiotensin IV (AT IV). There are conflicting data regarding their role in damage to the CNS in various diseases, including the coronavirus infection.The second participant in the pathogenesis of brain damage in COVID-19 is CD147 - the inducer of extracellular matrix metalloproteinases. This molecule is expressed on the endothelial cells of cerebral microvessels, as well as on leukocytes present in the brain during neuroinflammation. The CD147 molecule plays a significant role in maintaining the structural and functional integrity of the blood-brain barrier by controlling the basal membrane permeability and by mediating the astrocyte-endothelial interactions. Via the above mechanisms, an exposure to SARS-CoV-2 leads to direct damage to the neurovascular unit of the brain.

A human three-dimensional neural-perivascular 'assembloid' promotes astrocytic development and enables modeling of SARS-CoV-2 neuropathology.

Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.

The microglial NLRP3 inflammasome is involved in human SARS-CoV-2 cerebral pathogenicity: A report of three post-mortem cases.

We herein report, by using confocal immunofluorescence, the colocalization of the SARS-CoV-2 nucleocapsid within neurons, astrocytes, oligodendrocytes and microglia in three deceased COVID-19 cases, of between 78 and 85 years of age at death. The viral nucleocapsid was detected together with its ACE2 cell entry receptor, as well as the NLRP3 inflammasome in cerebral cortical tissues. It is noteworthy that NLRP3 was colocalized with CD68 + macrophages in the brain and lung of the deceased, suggesting the critical role of this type of inflammasome in SARS-CoV-2 lesions of the nervous system/lungs and supporting its potential role as a therapeutic target.

Expression of SARS-CoV-2-related receptors in cells of the neurovascular unit: implications for HIV-1 infection.

BACKGROUND: Neurological complications are common in patients affected by COVID-19 due to the ability of SARS-CoV-2 to infect brains. While the mechanisms of this process are not fully understood, it has been proposed that SARS-CoV-2 can infect the cells of the neurovascular unit (NVU), which form the blood-brain barrier (BBB). The aim of the current study was to analyze the expression pattern of the main SARS-CoV-2 receptors in naïve and HIV-1-infected cells of the NVU in order to elucidate a possible pathway of the virus entry into the brain and a potential modulatory impact of HIV-1 in this process. METHODS: The gene and protein expression profile of ACE2, TMPRSS2, ADAM17, BSG, DPP4, AGTR2, ANPEP, cathepsin B, and cathepsin L was assessed by qPCR, immunoblotting, and immunostaining, respectively. In addition, we investigated if brain endothelial cells can be affected by the exposure to the S1 subunit of the S protein, the domain responsible for the direct binding of SARS-CoV-2 to the ACE2 receptors. RESULTS: The receptors involved in SARS-CoV-2 infection are co-expressed in the cells of the NVU, especially in astrocytes and microglial cells. These receptors are functionally active as exposure of endothelial cells to the SARS CoV-2 S1 protein subunit altered the expression pattern of tight junction proteins, such as claudin-5 and ZO-1. Additionally, HIV-1 infection upregulated ACE2 and TMPRSS2 expression in brain astrocytes and microglia cells. CONCLUSIONS: These findings provide key insight into SARS-CoV-2 recognition by cells of the NVU and may help to develop possible treatment of CNS complications of COVID-19.