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.

The Blood-Brain Barrier in Health and Disease.

The blood-brain barrier (BBB) is a complex network of tightly regulated cells and transport proteins that separate the circulating blood from the brain tissue [...].

A year in review: brain barriers and brain fluids research in 2022.

This aim of this editorial is to highlight progress made in brain barrier and brain fluid research in 2022. It covers studies on the blood-brain, blood-retina and blood-CSF barriers (choroid plexus and meninges), signaling within the neurovascular unit and elements of the brain fluid systems. It further discusses how brain barriers and brain fluid systems are impacted in CNS diseases, their role in disease progression and progress being made in treating such diseases.

Nanomedicines for brain diseases: where we are and where we are going.

Graphical abstract [Formula: see text].

[The neurology of COVID-19]. / La neurología de COVID-19.

The clinical manifestations of COVID-19 are reminiscent of those of acute respiratory distress syndrome induced by cytokine release syndrome and secondary hemophagocytic lymphohistiocytosis that is observed in patients with other coronaviruses such as SARS-CoV and MERS-CoV. Neurologists face the challenge of assessing patients with pre-existing neurological diseases who have contracted SARS-CoV-2, patients with COVID-19 who present neurological emergencies, and patients who are carriers of the virus and have developed secondary neurological complications, either during the course of the disease or after it. Some authors and recent literature reports suggest that the presence of neurological manifestations in patients who are carriers of SARS-CoV-2 may be associated with a greater severity of the disease.

Las manifestaciones clínicas de COVID-19 recuerdan las del síndrome de insuficiencia respiratoria aguda inducido por el síndrome de liberación de citocinas y la linfohistiocitosis hemofagocitica observada en pacientes con otros coronavirus como SARS-CoV y MERS-CoV. Los neurólogos tienen el reto de evaluar pacientes con enfermedades neurológicas preexistentes que contraen SARS-CoV-2, pacientes con COVID-19 que presentan emergencias neurológicas y pacientes portadores del virus que desarrollan complicaciones neurológicas secundarias, durante el curso de la enfermedad o posterior a la misma. Algunos autores y reportes en la literatura recientes sugieren que las manifestaciones neurológicas en pacientes portadores de SARS-CoV-2 pueden asociarse con mayor gravedad de la enfermedad.

Brain gene therapy with Trojan horse lipid nanoparticles.

The COVID-19 mRNA vaccine was developed by the scalable manufacture of lipid nanoparticles (LNPs) that encapsulate mRNA within the lipid. There are many potential applications for this large nucleic acid delivery technology, including the delivery of plasmid DNA for gene therapy. However, gene therapy for the brain requires LNP delivery across the blood-brain barrier (BBB). It is proposed that LNPs could be reformulated for brain delivery by conjugation of receptor-specific monoclonal antibodies (MAbs) to the LNP surface. The MAb acts as a molecular Trojan horse to trigger receptor-mediated transcytosis (RMT) of the LNP across the BBB and subsequent localization to the nucleus for transcription of the therapeutic gene. Trojan horse LNPs could enable new approaches to gene therapy of the brain.

Blood-brain barrier penetration of non-replicating SARS-CoV-2 and S1 variants of concern induce neuroinflammation which is accentuated in a mouse model of Alzheimer's disease.

COVID-19 and especially Long COVID are associated with severe CNS symptoms and may place persons at risk to develop long-term cognitive impairments. Here, we show that two non-infective models of SARS-CoV-2 can cross the blood-brain barrier (BBB) and induce neuroinflammation, a major mechanism underpinning CNS and cognitive impairments, even in the absence of productive infection. The viral models cross the BBB by the mechanism of adsorptive transcytosis with the sugar N-acetylglucosamine being key. The delta and omicron variants cross the BB B faster than the other variants of concern, with peripheral tissue uptake rates also differing for the variants. Neuroinflammation induced by icv injection of S1 protein was greatly enhanced in young and especially in aged SAMP8 mice, a model of Alzheimer's disease, whereas sex and obesity had little effect.

Blood-brain barrier function in response to SARS-CoV-2 and its spike protein.

The typical manifestation of coronavirus 2 (CoV-2) infection is a severe acute respiratory syndrome (SARS) accompanied by pneumonia (COVID-19). However, SARS-CoV-2 can also affect the brain, causing chronic neurological symptoms, variously known as long, post, post-acute, or persistent COVID-19 condition, and affecting up to 40% of patients. The symptoms (fatigue, dizziness, headache, sleep disorders, malaise, disturbances of memory and mood) usually are mild and resolve spontaneously. However, some patients develop acute and fatal complications, including stroke or encephalopathy. Damage to the brain vessels mediated by the coronavirus spike protein (S-protein) and overactive immune responses have been identified as leading causes of this condition. However, the molecular mechanism by which the virus affects the brain still needs to be fully delineated. In this review article, we focus on interactions between host molecules and S-protein as the mechanism allowing the transit of SARS-CoV-2 through the blood-brain barrier to reach the brain structures. In addition, we discuss the impact of S-protein mutations and the involvement of other cellular factors conditioning the pathophysiology of SARS-CoV-2 infection. Finally, we review current and future COVID-19 treatment options.

Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Implementation of blood-brain barrier on microfluidic chip: Recent advance and future prospects.

The complex structure of the blood-brain barrier (BBB) hinders its modeling and the treatment of brain diseases. The microfluidic technology promotes the development of BBB-on-a-chip platforms, which can be used to reproduce the complex brain microenvironment and physiological reactions. Compared with traditional transwell technology, microfluidic BBB-on-a-chip shows great technical advantages in terms of flexible control of fluid shear stress in the chip and fabrication efficiency of the chip system, which can be enhanced by the development of lithography and three-dimensional (3D) printing. It is convenient to accurately monitor the dynamic changes of biochemical parameters of individual cells in the model by integrating an automatic super-resolution imaging sensing platform. In addition, biomaterials, especially hydrogels and conductive polymers, solve the limitations of microfluidic BBB-on-a-chip by compounding onto microfluidic chip to provide a 3D space and special performance on the microfluidic chip. The microfluidic BBB-on-a-chip promotes the development of basic research, including cell migration, mechanism exploration of neurodegenerative diseases, drug barrier permeability, SARS-CoV-2 pathology. This study summarizes the recent advances, challenges and future prospects of microfluidic BBB-on-a-chip, which can help to promote the development of personalized medicine and drug discovery.

Recombinant human plasma gelsolin reverses increased permeability of the blood-brain barrier induced by the spike protein of the SARS-CoV-2 virus.

BACKGROUND: Plasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood-brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit). MATERIALS AND METHODS: Two- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response. RESULTS: pGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5). CONCLUSION: Due to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19.

Citicoline and COVID-19: vis-à-vis conjectured.

Coronavirus disease 2019 (COVID-19) is a current pandemic disease caused by a novel severe acute respiratory syndrome coronavirus virus respiratory type 2 (SARS-CoV-2). SARS-CoV-2 infection is linked with various neurological manifestations due to cytokine-induced disruption of the blood brain barrier (BBB), neuroinflammation, and peripheral neuronal injury, or due to direct SARS-CoV-2 neurotropism. Of note, many repurposed agents were included in different therapeutic protocols in the management of COVID-19. These agents did not produce an effective therapeutic eradication of SARS-CoV-2, and continuing searching for novel anti-SARS-CoV-2 agents is a type of challenge nowadays. Therefore, this study aimed to review the potential anti-inflammatory and antioxidant effects of citicoline in the management of COVID-19.

The Effects of Viruses on Insulin Sensitivity and Blood-Brain Barrier Function.

In this review manuscript, we discuss the effects of select common viruses on insulin sensitivity and blood-brain barrier (BBB) function and the potential overlapping and distinct mechanisms involved in these effects. More specifically, we discuss the effects of human immunodeficiency virus (HIV), herpes, hepatitis, influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 viruses on insulin sensitivity and BBB function and the proposed underlying mechanisms. These viruses differ in their ability to be transported across the BBB, disrupt the BBB, and/or alter the function of the BBB. For RSV and SARS-CoV-2, diabetes increases the risk of infection with the virus, in addition to viral infection increasing the risk for development of diabetes. For HIV and hepatitis C and E, enhanced TNF-a levels play a role in the detrimental effects. The winter of 2022-2023 has been labeled as a tridemic as influenza, RSV, and COVID-19 are all of concern during this flu season. There is an ongoing discussion about whether combined viral exposures of influenza, RSV, and COVID-19 have additive, synergistic, or interference effects. Therefore, increased efforts are warranted to determine how combined viral exposures affect insulin sensitivity and BBB function.

Deconstructing the functional neuroanatomy of the choroid plexus: an ontogenetic perspective for studying neurodevelopmental and neuropsychiatric disorders.

The choroid plexus (CP) is a delicate and highly vascularized structure in the brain comprised of a dense network of fenestrated capillary loops that help in the synthesis, secretion and circulation of cerebrospinal fluid (CSF). This unique neuroanatomical structure is comprised of arachnoid villi stemming from frond-like surface projections-that protrude into the lumen of the four cerebral ventricles-providing a key source of nutrients to the brain parenchyma in addition to serving as a 'sink' for central nervous system metabolic waste. In fact, the functions of the CP are often described as being analogous to those of the liver and kidney. Beyond forming a barrier/interface between the blood and CSF compartments, the CP has been identified as a modulator of leukocyte trafficking, inflammation, cognition, circadian rhythm and the gut brain-axis. In recent years, advances in molecular biology techniques and neuroimaging along with the use of sophisticated animal models have played an integral role in shaping our understanding of how the CP-CSF system changes in relation to the maturation of neural circuits during critical periods of brain development. In this article we provide an ontogenetic perspective of the CP and review the experimental evidence implicating this structure in the pathophysiology of neurodevelopmental and neuropsychiatric disorders.

Markers of blood-brain barrier disruption increase early and persistently in COVID-19 patients with neurological manifestations.

Background: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is associated with disorders affecting the peripheral and the central nervous system. A high number of patients develop post-COVID-19 syndrome with the persistence of a large spectrum of symptoms, including neurological, beyond 4 weeks after infection. Several potential mechanisms in the acute phase have been hypothesized, including damage of the blood-brain-barrier (BBB). We tested weather markers of BBB damage in association with markers of brain injury and systemic inflammation may help in identifying a blood signature for disease severity and neurological complications. Methods: Blood biomarkers of BBB disruption (MMP-9, GFAP), neuronal damage (NFL) and systemic inflammation (PPIA, IL-10, TNFα) were measured in two COVID-19 patient cohorts with high disease severity (ICUCovid; n=79) and with neurological complications (NeuroCovid; n=78), and in two control groups free from COVID-19 history, healthy subjects (n=20) and patients with amyotrophic lateral sclerosis (ALS; n=51). Samples from COVID-19 patients were collected during the first and the second wave of COVID-19 pandemic in Lombardy, Italy. Evaluations were done at acute and chronic phases of the COVID-19 infection. Results: Blood biomarkers of BBB disruption and neuronal damage are high in COVID-19 patients with levels similar to or higher than ALS. NeuroCovid patients display lower levels of the cytokine storm inducer PPIA but higher levels of MMP-9 than ICUCovid patients. There was evidence of different temporal dynamics in ICUCovid compared to NeuroCovid patients with PPIA and IL-10 showing the highest levels in ICUCovid patients at acute phase. On the contrary, MMP-9 was higher at acute phase in NeuroCovid patients, with a severity dependency in the long-term. We also found a clear severity dependency of NFL and GFAP levels, with deceased patients showing the highest levels. Discussion: The overall picture points to an increased risk for neurological complications in association with high levels of biomarkers of BBB disruption. Our observations may provide hints for therapeutic approaches mitigating BBB disruption to reduce the neurological damage in the acute phase and potential dysfunction in the long-term.

COVID-19: a new emerging respiratory disease from the neurological perspective.

Coronavirus disease 2019 (COVID-19) has become a challenging public health catastrophe worldwide. The newly emerged disease spread in almost all countries and infected 100 million persons worldwide. The infection is not limited to the respiratory system but involves various body systems and may lead to multiple organ failure. Tissue degenerative changes result from direct viral invasion, indirect consequences, or through an uncontrolled immune response. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads to the brain via hematogenous and neural routes accompanied with dysfunction of the blood-brain barrier. The involvement of the central nervous system is now suspected to be among the main causes of death. The present review discusses the historical background of coronaviruses, their role in previous and ongoing pandemics, the way they escape the immune system, why they are able to spread despite all undertaken measures, in addition to the neurological manifestations, long-term consequences of the disease, and various routes of viral introduction to the CNS.

The Adverse Outcome Pathway Framework Applied to Neurological Symptoms of COVID-19.

Several reports have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to also be neurotropic. However, the mechanisms by which SARS-CoV-2 induces neurologic injury, including neurological and/or psychological symptoms, remain unclear. In this review, the available knowledge on the neurobiological mechanisms underlying COVID-19 was organized using the AOP framework. Four AOPs leading to neurological adverse outcomes (AO), anosmia, encephalitis, stroke, and seizure, were developed. Biological key events (KEs) identified to induce these AOs included binding to ACE2, blood-brain barrier (BBB) disruption, hypoxia, neuroinflammation, and oxidative stress. The modularity of AOPs allows the construction of AOP networks to visualize core pathways and recognize neuroinflammation and BBB disruption as shared mechanisms. Furthermore, the impact on the neurological AOPs of COVID-19 by modulating and multiscale factors such as age, psychological stress, nutrition, poverty, and food insecurity was discussed. Organizing the existing knowledge along an AOP framework can represent a valuable tool to understand disease mechanisms and identify data gaps and potentially contribute to treatment, and prevention. This AOP-aligned approach also facilitates synergy between experts from different backgrounds, while the fast-evolving and disruptive nature of COVID-19 emphasizes the need for interdisciplinarity and cross-community research.

COVID-19 and neurodegeneration: The mitochondrial connection.

There is still a significant lack of knowledge regarding many aspects of the etiopathology and consequences of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. For example, the variety of molecular mechanisms mediating this infection, and the long-term consequences of the disease remain poorly understood. It first seemed like the SARS-CoV-2 infection primarily caused a serious respiratory syndrome. However, over the last years, an increasing number of studies also pointed towards the damaging effects of this infection has on the central nervous system (CNS). In fact, evidence suggests a possible disruption of the blood-brain barrier and deleterious effects on the CNS, especially in patients who already suffer from other pathologies, such as neurodegenerative disorders. The molecular mechanisms behind these effects on the CNS could involve the dysregulation of mitochondrial physiology, a well-known early marker of neurodegeneration and a hallmark of aging. Moreover, mitochondria are involved in the activation of the inflammatory response, which has also been broadly described in the CNS in COVID-19. Here, we critically review the current bibliography regarding the presence of neurodegenerative symptoms in COVID-19 patients, with a special emphasis on the mitochondrial mechanisms of these disorders.

Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients.

Neurofilament light chain (NfL) is a specific biomarker of neuro-axonal damage. Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in blood-brain barrier (BBB) integrity. We explored neuro-axonal damage, alteration of BBB integrity and SARS-CoV-2 RNA presence in COVID-19 patients with severe neurological symptoms (neuro-COVID) as well as neuro-axonal damage in COVID-19 patients without severe neurological symptoms according to disease severity and after recovery, comparing the obtained findings with healthy donors (HD). Overall, COVID-19 patients (n = 55) showed higher plasma NfL levels compared to HD (n = 31) (p < 0.0001), especially those who developed ARDS (n = 28) (p = 0.0005). After recovery, plasma NfL levels were still higher in ARDS patients compared to HD (p = 0.0037). In neuro-COVID patients (n = 12), higher CSF and plasma NfL, and CSF MMP-2 levels in ARDS than non-ARDS group were observed (p = 0.0357, p = 0.0346 and p = 0.0303, respectively). SARS-CoV-2 RNA was detected in four CSF and two plasma samples. SARS-CoV-2 RNA detection was not associated to increased CSF NfL and MMP levels. During COVID-19, ARDS could be associated to CNS damage and alteration of BBB integrity in the absence of SARS-CoV-2 RNA detection in CSF or blood. CNS damage was still detectable after discharge in blood of COVID-19 patients who developed ARDS during hospitalization.