Patients with coronavirus disease 2019 (COVID-19) have been frequently reported to exhibit neurological manifestations and disruption of the blood-brain barrier (BBB). Among the risk factors for BBB breakdown, the loss of endothelial cells and pericytes has caused widespread concern. Recent studies have revealed that severe acute respiratory syndrome coronavirus 2 envelope (S2E) protein caused cell death. We tested the hypothesis that the S2E protein alone could induce BBB dysfunction. The S2E protein bound to human BBB-related cells and inhibited cell viability in a dose- and time-dependent manner. Importantly, the S2E protein disrupted barrier function in an in vitro BBB model composed of HCMEC/D3 (brain endothelial cell line), HBVP (brain vascular pericyte), and U87MG (astrocyte cell line) cells and suppressed the expression of major genes involved in maintaining endothelial permeability and function. In addition, the S2E protein crossed the HCMEC/D3 monolayer. The S2E protein triggered inflammatory responses in HCMEC/D3 and U87MG cells. Taken together, these results show for the first time that the S2E protein has a negative impact on the BBB. Therapies targeting the S2E protein could protect against and treat central nervous system manifestations in COVID-19 patients. 639 frontcellneuro Frontiers in Cellular Neuroscience Front Cell Neurosci Frontiers Media SA PMC9445123 9445123 9445123 36082238 10.3389/fncel.2022.897564 The SARS-CoV-2 envelope protein disrupts barrier function in an in vitro human blood-brain barrier model Ju Jiahang 1 2 † Su Yuwen 1 2 † Zhou You 1 2 Wei Hui 1 2 Xu Qi 1 2 * 1 State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China 2 Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China Edited by: Helena Solleiro-Villavicencio, Universidad Autónoma de la Ciudad de México, Mexico Reviewed by: María Del Carmen Cárdenas-Aguayo, Universidad Nacional Autónoma de México, Mexico; Ashutosh Kumar, All India Institute of Medical Sciences (Patna), India ✉ *Correspondence: Qi Xu xuqi@pumc.edu.cn This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience †These authors have contributed equally to this work 23 8 2022 16 897564 897564 7 9 2022 Copyright © 2022 Ju, Su, Zhou, Wei and Xu. One possibility is that SARS-CoV-2 invades the brain via the olfactory nerves due to the presence of viral particles at the neural-mucosal interface in the olfactory mucosa in autopsied patient tissues (Meinhardt et al., 2021 ). Alternatively, SARS-CoV-2 may cross the blood-brain barrier (BBB), as evidenced by the detection of viral RNA in cerebrospinal fluid (Moriguchi et al., 2020 ). In human brain organoid models, SARS-CoV-2 can directly infect neurons and choroid plexus epithelial cells (Jacob et al., 2020 ). The present study aimed to assess whether the SARS-CoV-2 envelope protein negatively affects BBB functions and induces neuroinflammatory effects in an in vitro model of the BBB. Materials and methods Cell culture and treatment Human immortalized cerebral microvascular endothelial cells (HCMEC/D3), human brain vascular pericytes (HBVP), and human glioblastoma cells (U87MG) were obtained from Xinyu Biotechnology Co., Ltd (Shanghai, China). All cells were maintained in a humidified incubator (37°C, 5% CO 2 ). SARS-CoV-2 envelope protein crosses the BBB and disrupts barrier integrity and permeability To evaluate the effects of the S2E protein on BBB functions, an in vitro Transwell barrier BBB model was established, in which HCMEC/D3 cells on the luminal side served as brain endothelial cells, while HBVP cells and U87MG cells on the underside of the insert served as brain vascular pericytes and astrocytes, respectively ( Figure 2A ). The S2E protein was administered on the luminal side. Then, BBB integrity was assessed by TEER, and BBB permeability was assessed by the transmissivity of 10 kDa FITC-dextran. Taken together, our data strongly suggest that the S2E protein has the potential to cause BBB leakage. Figure 3 SARS-CoV-2 envelope protein impairs endothelial cell barrier function. (A) Relative mRNA levels of ZO-1, PECAM1, PGP, and SLC2A1 in HCMEC/D3 cells, as determined by RT-PCR. (B) Immunofluorescence images of ZO-1 in HCMEC/D3 cells. Scale bar = 10 μm. The data shown are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001. Furthermore, treating both cell lines with S2E protein, ELISA assay showed markedly increases only in The possible mechanism by which the S2E protein crosses the BBB is shown in Figure 5 . During infection, the envelope protein can bind to brain endothelial cells and traverse the BBB, leading to damaging the BBB and inducing inflammatory responses in astrocytes. In addition, the inflammatory factors produced by brain endothelial cells and astrocytes, in turn, may exacerbate BBB damage. Figure 5 A schematic diagram depicting the potential mechanism by which the SARS-CoV-2 envelope protein crosses the BBB. The S2E protein disrupts BBB integrity, leading to inflammation in endothelial cells and astrocytes. This study has several limitations. Glossary Abbreviations COVID-19 coronavirus disease 2019 BBB blood-brain barrier SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 S2S SARS-CoV-2 spike S2E SARS-CoV-2 envelope CNS central nervous system ARDS alone causes acute respiratory distress syndrome TLR2 toll-like receptor 2 MHC-I MHC class I ER endoplasmic reticulum HAND HIV-associated neurocognitive disorder. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel.2022.897564/full#supplementary-material Click here for additional data file. References Associated Data Supplementary Materials Click here for additional data file.

Objective To investigate the association between blood–brain barrier permeability, brain metabolites, microstructural integrity of the white matter, and cognitive impairment (CI) in post-acute sequelae of SARS-COV-2 infection (PASC). Methods In this multimodal longitudinal MRI study 14 PASC participants with CI and 10 healthy controls were enrolled. All completed investigations at 3 months following acute infection (3 months ± 2 weeks SD), and 10 PASC participants completed at 12 months ± 2.22 SD weeks. The assessments included a standard neurological assessment, a cognitive screen using the brief CogState battery and multi-modal MRI derived metrics from Dynamic contrast enhanced (DCE) perfusion Imaging, Diffusion Tensor Imaging (DTI), and single voxel proton Magnetic Resonance Spectroscopy. These measures were compared between patients and controls and correlated with cognitive scores. Results At baseline, and relative to controls, PASC participants had higher K-Trans and Myo-inositol, and lower levels of Glutamate/Glutamine in the frontal white matter (FWM) (p < 0.01) as well as in brain stem (p < 0.05), and higher FA and lower MD in the FWM (p < 0.05). In PASC participants, FA and MD decreased in the FWM at 12 months compared to baseline (p < 0.05). K-Trans and metabolite concentrations did not change significantly over time. Neurocognitive scores did not correlation with the increased permeability (K trans). Interpretation PASC with CI is associated with BBB impairment, loss of WM integrity, and inflammation at 3 months which significantly but not uniformly improved at 12 months. The loss of WM integrity is possibly mediated by BBB impairment and associated glutamatergic excitotoxicity. 1401 frontneurology Frontiers in Neurology Front Neurol Frontiers Media SA PMC11097901 11097901 11097901 38756214 10.3389/fneur.2024.1350848 Blood brain barrier disruption and glutamatergic excitotoxicity in post-acute sequelae of SARS COV-2 infection cognitive impairment: potential biomarkers and a window into pathogenesis Chaganti Joga 1 * Poudel Govinda 2 Cysique Lucette Adeline 3 Dore Gregory J 4 Kelleher Anthony 4 5 Matthews Gael 4 Darley David 3 Byrne Anthony 5 Jakabek David 5 Zhang Xin 6 Lewis Marrissa 5 Jha Nikhil 7 Brew Bruce James 3 8 1 Thomas Jefferson University, Philadelphia, PA, United States 2 Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, VIC, Australia 3 Department of Neurology and Immunology, Peter Duncan Neuroscience Unit, St Vincent’s Hospital, University of New South Wales, Darlinghurst, NSW, Australia 4 The Kirby Institute, Faculty of Medicine, University of New South Wales, Kensington, NSW, Australia 5 St Vincent’s Hospital, University of NSW, Darlinghurst, NSW, Australia 6 Royal Prince Alfred Hospital, Sydney, NSW, Australia 7 The Canberra Hospital, Canberra, ACT, Australia 8 University of Notre Dame, Sydney, NSW, Australia Edited by: Alina Gonzalez-Quevedo, Instituto de Neurología y Neurocirugía, La Habana, Cuba Reviewed by: Wakiro Sato, National Center of Neurology and Psychiatry, Japan Sergio Gonzalez-Garcia, University of Havana, Cuba ✉ *Correspondence: Joga Chaganti, joga.chaganti@jefferson.edu 2 5 2024 15 1350848 1350848 16 5 2024 Copyright © 2024 Chaganti, Poudel, Cysique, Dore, Kelleher, Matthews, Darley, Byrne, Jakabek, Zhang, Lewis, Jha and Brew. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Abstract Objective To investigate the association between blood–brain barrier permeability, brain metabolites, microstructural integrity of the white matter, and cognitive impairment (CI) in post-acute sequelae of SARS-COV-2 infection (PASC). We believe this is the first longitudinal study to show that there is blood brain barrier disruption associated with glutamatergic excitotoxicity that is likely responsible for the neurocognitive impairment. Our findings support further studies of these MRI techniques as potential biomarkers for PASC CI to facilitate diagnosis, appropriate timing of potential therapy and its monitoring. Introduction Cognitive impairment (CI) is one of the common symptoms of post-acute SARS CoV-2 infection (PASC), otherwise known as long covid ( 1 ). The pathogenesis, however, remains unclear with possibilities ranging from immune dysregulation, autoimmunity, microthrombi, and blood–brain barrier (BBB) impairment ( 2 , 3 ). To date, there is only one study which explored the role of BBB disruption using MR imaging, but the study did not have a longitudinal design ( 4 ). Brain imaging has provided a limited contribution to understanding of CI development following COVID-19. Studies using standard MRI brain scan sequences have mostly focused on severely affected patients during acute infection showing ischemia, hemorrhage, venous sinus thromboses ( 2 , 5 , 6 ). Seven of those participants had been enrolled into the ADAPT study, a prospective cohort of 128 SARS-CoV-2 positive patients and had received serial measurements of cognition with the Cogstate Brief Battery ( 1 ). In brief, the neurocognitive score (NCS) was derived from CogState: individual task z -scores were averaged into a mean z -score (higher equates to a better objective global performance) corrected for normative age, education and sex effects. Individuals with a prior history of drug use, significant head injury, psychiatric illness, and hepatitis C virus co-infection were excluded. Figure 5 Correlation of K trans with NCS (A) Trend-level correlation between brainstem K-Trans and NCS and (B) statistically significant correlation of k trans and FA in the

Endothelial dysfunction plays a central role in COVID-19 pathogenesis, by affecting vascular homeostasis and worsening thromboinflammation. This imbalance may contribute to blood–brain barrier (BBB) disruption, which has been reported in long COVID-19 patients with neurological sequelae. The kallikrein–kinin system (KKS) generates bradykinin (BK), a proinflammatory peptide that induces microvascular leakage via B2R. Under inflammatory conditions, BK is converted to Des-Arg-BK (DABK), which activates B1R, a receptor upregulated in inflamed tissues. DABK is degraded by ACE2, the main SARS-CoV-2 receptor; thus, viral binding and ACE2 downregulation may lead to DABK/B1R imbalance. Here, we investigated these interactions using human brain microvascular endothelial cells (HBMECs), as a model of the BBB. Since endothelial cell lines express low levels of ACE2, HBMECs were modified with an ACE2-carrying pseudovirus. SARS-CoV-2 replication was confirmed by RNA, protein expression, and infectious particles release. Infection upregulated cytokines and endothelial permeability, enhancing viral and leukocyte transmigration. Additionally, viral replication impaired ACE2 function in HBMECs, amplifying the response to DABK, increasing nitric oxide (NO) production, and further disrupting endothelial integrity. Our findings reveal a mechanism by which SARS-CoV-2 impacts the BBB and highlights the ACE2/KKS/B1R axis as a potential contributor to long COVID-19 neurological symptoms.

Background The brain vasculature is a key player in neurological manifestations of COVID-19. Infection of brain endothelial cells with SARS-CoV-2 along with circulating cytokines may cause dysfunction of the blood-brain barrier (BBB). Solute carrier transporters (SLCs) in brain endothelial cells regulate substrate transport across the BBB. Here, it was hypothesized that transport functions of SLCs will be impaired by interactions with viral proteins, and subsequently, data-mining studies were performed. Methods Virus-host protein-protein interaction data for SARS-CoV-2 infection were retrieved from the BioGRID database, filtered for SLCs, and then annotated for relevant expression in brain endothelial cells using a mouse brain transcriptomics database. Host SLCs expressed in brain endothelial cells were further explored using publicly available databases and information in the literature. Functional Annotation Clustering was performed using DAVID, and Enrichr served for pathway analysis. Substrates were retrieved from NCBI Gene. Links to monogenic disorders were retrieved from Online Mendelian Inheritance in Man™ and screened for disorders of the nervous system. Interactome data for viral proteins of SARS-CoV-2 were retrieved from BioGRID. Reports for host SLCs in viral receptor functions, viral entry mechanisms, and other major roles in the viral cycle were explored in databases (VThunter) and literature. ATP-binding cassette transporters (ABCs) were studied in parallel. Results N = 80 host SLCs showed relevant expression in brain endothelial cells whereby amino acid transporter stood out. N = 24/80 host SLCs were linked to monogenic disorders of the nervous system. N = 9/29 SARS-CoV-2 viral proteins had strong links to SLCs and key functions in viral infection (e.g., interferon response). SLCs serving as viral receptors and with closely associated functions were significantly enriched among all known listed viral receptors (chi-square test, p = 0.001). Literature searches for host SLCs revealed involvement of a subset of SLCs in infection mechanisms for SARS-CoV-2 and more broadly for other viruses. N = 17 host ABCs were found in brain endothelial cells where they may serve as efflux transporters. Discussion This hypothesis-generating work proposes a set of N = 80 host SLCs expressed in endothelial cells as contributors to BBB impairment after SARS-CoV-2 infection. Theoretically, persistent dysfunction of SLCs at the BBB, in particular insufficient transport of amino acids, could be one of many reasons for cognitive changes in long-COVID. Functions of SLCs in viral entry and associated roles deserve close attention.

SARS-CoV-2 infection has been implicated in hippocampal damage, contributing to the pathogenesis of dysexecutive syndrome observed in post-COVID-19 patients. Given the growing prevalence of long-COVID worldwide, understanding how SARS-CoV-2 affects hippocampal structure and function has become an urgent scientific and clinical priority. The hippocampus-crucial for memory, emotional regulation, and executive functioning-is especially susceptible to viral-driven neuroinflammatory cascades. SARS-CoV-2 triggers astrocyte and microglia activation, disrupts blood-brain barrier integrity, and induces cytokine-mediated neurotoxicity, ultimately impairing neuroplasticity and neurogenesis. These mechanisms converge to produce cognitive and affective disturbances-most notably fatigue, apathy, low mood, and executive dysfunction-that typify dysexecutive syndrome in long-COVID. This review synthesizes current evidence from clinical and experimental studies, integrating findings on viral neurotropism, hippocampal hypometabolism, and astrocyte-mediated neurodegeneration. Distinctions between depressive symptoms driven by neuroinflammation and classical depressive disorders are clarified to improve diagnostic accuracy and guide personalized treatment. Emerging data on the neuroprotective role of COVID-19 vaccination-particularly its capacity to modulate microglial activation and support hippocampal neurogenesis-are also examined. Overall, the findings underscore the need for targeted therape