Ketone bodies supplementation restores the barrier function, induces a metabolic switch, and elicits beta-hydroxybutyrate diffusion across a monolayer of iPSC-derived brain microvascular endothelial cells.

Glucose constitutes the main source of energy for the central nervous system (CNS), its entry occurring at the blood-brain barrier (BBB) via the presence of glucose transporter 1 (GLUT1). However, under food intake restrictions, the CNS can utilize ketone bodies (KB) as an alternative source of energy. Notably, the relationship between the BBB and KBs and its effect on their glucose metabolism remains poorly understood. In this study, we investigated the effect of glucose deprivation on the brain endothelium in vitro, and supplementation with KBs using induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cell-like cells (iBMECs). Glucose-free environment significantly decreased cell metabolic activity and negatively impacted the barrier function. In addition, glucose deprivation did not increase GLUT1 expression but also resulted in a decrease in glucose uptake and glycolysis. Supplementation of glucose-deprived iBMECs monolayers with KB showed no improvement and even worsened upon treatment with acetoacetate. However, under a hypoglycemic condition in the presence of KBs, we noted a slight improvement of the barrier function, with no changes in glucose uptake. Notably, hypoglycemia and/or KB pre-treatment elicited a saturable beta-hydroxybutyrate diffusion across iBMECs monolayers, such diffusion occurred partially via an MCT1-dependent mechanism. Taken together, our study highlights the importance of glucose metabolism and the reliance of the brain endothelium on glucose and glycolysis for its function, such dependence is unlikely to be covered by KBs supplementation. In addition, KB diffusion at the BBB appeared induced by KB pre-treatment and appears to involve an MCT1-dependent mechanism.

An in vitro model of glucose transporter 1 deficiency syndrome at the blood-brain barrier using induced pluripotent stem cells.

Glucose is an important source of energy for the central nervous system. Its uptake at the blood-brain barrier (BBB) is mostly mediated via glucose transporter 1 (GLUT1), a facilitated transporter encoded by the SLC2A1 gene. GLUT1 Deficiency Syndrome (GLUT1DS) is a haploinsufficiency characterized by mutations in the SLC2A1 gene, resulting in impaired glucose uptake at the BBB and clinically characterized by epileptic seizures and movement disorder. A major limitation is an absence of in vitro models of the BBB reproducing the disease. This study aimed to characterize an in vitro model of GLUT1DS using human pluripotent stem cells (iPSCs). Two GLUT1DS clones were generated (GLUT1-iPSC) from their original parental clone iPS(IMR90)-c4 by CRISPR/Cas9 and differentiated into brain microvascular endothelial cells (iBMECs). Cells were characterized in terms of SLC2A1 expression, changes in the barrier function, glucose uptake and metabolism, and angiogenesis. GLUT1DS iPSCs and iBMECs showed comparable phenotype to their parental control, with exception of reduced GLUT1 expression at the protein level. Although no major disruption in the barrier function was reported in the two clones, a significant reduction in glucose uptake accompanied by an increase in glycolysis and mitochondrial respiration was reported in both GLUT1DS-iBMECs. Finally, impaired angiogenic features were reported in such clones compared to the parental clone. Our study provides the first documented characterization of GLUT1DS-iBMECs generated by CRISPR-Cas9, suggesting that GLUT1 truncation appears detrimental to brain angiogenesis and brain endothelial bioenergetics, but maybe not be detrimental to iBMECs differentiation and barriergenesis. Our future direction is to further characterize the functional outcome of such truncated product, as well as its impact on other cells of the neurovascular unit.

In Vitro Models of the Human Blood-Brain Barrier Utilising Human Induced Pluripotent Stem Cells: Opportunities and Challenges.

The blood-brain barrier (BBB) is a component of the neurovascular unit formed by specialized brain microvascular endothelial cells surrounded by astrocytes end-feet processes, pericytes, and a basement membrane. The BBB plays an important role in the maintenance of brain homeostasis and has seen a growing involvement in the pathophysiology of various neurological diseases. On the other hand, the presence of such a barrier remains an important challenge for drug delivery to treat such illnesses.Since the pioneering work describing the isolation and cultivation of primary brain microvascular cells about 50 years ago until now, the development of an in vitro model of the BBB that is scalable, capable to form tight monolayers, and predictive of drug permeability in vivo remained extremely challenging.The recent description of the use of induced pluripotent stem cells (iPSCs) as a modeling tool for neurological diseases raised momentum into the use of such cells to develop new in vitro models of the BBB. This chapter will provide an exhaustive description of the use of iPSCs as a source of cells for modeling the BBB in vitro, describe the advantages and limitations of such model, as well as describe their prospective use for disease modeling and drug permeability screening platforms.

Abeta peptides disrupt the barrier integrity and glucose metabolism of human induced pluripotent stem cell-derived brain microvascular endothelial cells.

Amyloid ß (Aß) peptides are key components of Alzheimer's disease and cerebral amyloid angiopathy and have been associated with detrimental effects at the blood-brain barrier (BBB) in vivo. Yet, the cellular and molecular mechanisms by which such peptides exert their effect on the brain vasculature remain unclear. This study aimed to assess the cellular response of induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) to Aß peptides. Changes in the barrier function, efflux transporters activity, glucose uptake, and metabolism were assessed in such model. Although iPSC-derived BMECs sustained prolonged exposure (<72 h) to a high level of Aß peptides including Aß42, such cells also suffered from a loss of barrier integrity, coupled with reduced glucose uptake and impaired bioenergetic activity. Taken together, this study shows the ability of iPSC-derived BMECs to reproduce features observed in other models and suggests that Aß peptides may compromise the BBB via different targets.

Investigations on Synergistic and Antioxidant Actions of Medicinal Plant- Based Biosynthesis of Zinc Oxide Nanoparticles Against E.coli and K. pneumonia Bacteria.

INTRODUCTION: Bacterial resistance to multiple drugs is increasing at an alarming rate in the current era and nanotechnology is one of the effective and novel approaches to overcome drug resistance. METHODS: Zinc Oxide Nanoparticles (ZnO NPs) have stronger antibacterial activity and are regarded as bio-safe nanomaterial. The aim of the present study is to synthesize the ZnO NPs using Aloe vera leaves extract and to investigate the synergistic effects and antioxidant actions of biosynthesized ZnO NPs against gram-negative bacteria E.coli and K. pneumoniae. The synergistic effect of ß-lactam antibiotics (meropenem and ciprofloxacin) was tested along with ZnO NPs using Kirby's disc diffusion assay. The antioxidant activity was investigated by α, α-diphenyl-ß- picrylhydrazyl (DPPH) method. RESULTS: Results of the study revealed that the antibacterial activity of the selected antibiotics was much enhanced by ZnO NPs than the antibiotics alone. The resistant antibiotic (ciprofloxacin) became sensitive when combined with ZnO NPs. The antioxidant activity reveals that biosynthesized ZnO NPs possess significantly higher (p<0.05) antioxidant activity (77%). CONCLUSION: The findings reveal that biosynthesized ZnO NPs have a much more eco-friendly approach. It can act as a strong potentiator of ß-lactam antibiotics and put forward the possibility to use them effectively in targeted drug delivery, pharmaceuticals and biomedical fields.

Neurolysin substrates bradykinin, neurotensin and substance P enhance brain microvascular permeability in a human in vitro model.

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of several acute neurodegenerative disorders, including stroke. Numerous studies suggest the involvement of bradykinin (BK), neurotensin (NT) and substance P (SP) in BBB impairment and oedema formation after stroke; however, there is paucity of data in regard to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The present study aimed to evaluate the direct effects of BK, NT and SP on the permeability of BBB in an in vitro model based on human induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. The combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model indicating potentiation of their action. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors. Additional mechanistic studies indicate that the short-term effects of these peptides are not mediated through alteration of tight-junction proteins claudin-5 and occludin, but likely involve redistribution of F-actin and secretion of vascular endothelial growth factor. This is the first experimental study to document the increased permeability of the BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognised actions of BK on BBB. Lastly, we demonstrate that peptidase neurolysin can neutralise the effects of these peptides on BBB, suggesting potential therapeutic implications.

Epileptic seizures.

Epilepsy is a condition marked by abnormal neuronal discharges or hyperexcitability of neurons with synchronicity and is recognized as a major public health concern. The pathology is categorized into three subgroups: acquired, idiopathic, and epilepsy of genetic or developmental origin. There are approximately 1000 associated genes and the role of γ-aminobutyric acid (GABA) mediated inhibition, as well as glutamate mediated excitation, forms the basis of pathology. Epilepsy is further classified as being of focal, general or unknown onset. Genetic predisposition, comorbidities and novel biomarkers are useful for prediction. Prevalent postictal symptoms are postictal headache and migraine, postictal psychosis and delirium, postictal Todd's paresis and postictal automatisms. Diagnostic methods include electroencephalography (EEG), computed tomography scan, magnetic resonance imaging (MRI), positron emission tomography, single photon emission computed tomography and genetic testing; EEG and MRI are the two main techniques. Clinical history and witness testimonies combined with a knowledge of seizure semiology helps in distinguishing between seizures. Clinical information and patient history do not always lead to a clear diagnosis, in which case EEG and 24-hour EEG monitoring with video recording (video-EEG/vEEG) help in seizure differentiation. Treatment includes first aid, therapeutics such as anti-epileptic drugs, surgery, ketogenic diet and gene therapy. In this review, we are focusing on summarizing published literature on epilepsy and epileptic seizures, and concisely apprise the reader of the latest cutting-edge advances and knowledge on epileptic seizures.