Membrane Complex Research Articles

Retinal and Choriocapillaris Thickness Changes in Spontaneously Diabetic Macaques.

Beyond the clinically apparent damage to the retinal vasculature, diabetes affects the neuroretina and choroid. Nonhuman primates (NHPs) serve as valuable models for human retinal diseases, including diabetic retinopathy. This study aimed to investigate changes in the thickness of the retina, RPE, and choriocapillaris in spontaneously diabetic cynomolgus macaques. Optical coherence tomography (OCT) images were obtained from 25 diabetic macaques and 26 age-matched nondiabetic macaques. The thickness of individual retinal layers in the macula, along with RPE and choriocapillaris, was measured manually. Choriocapillaris thickness was assessed in postmortem human globes from diabetic and nondiabetic individuals. Diabetes predominantly affected the inner retina more than the outer retina in macaques. Notably, the nasal retina showed greater thinning compared to the temporal retina. A slight but significant thickening of the inner nuclear layer was observed. No changes were detected in the thickness of RPE/Bruch's membrane complex via OCT. However, a significant increase in choriocapillaris thickness was noted in the diabetic macaques. Postmortem human specimens from patients with nonproliferative diabetic retinopathy (NPDR) also demonstrated thickening of choriocapillaris/Bruch's membrane. Consistent with humans, early diabetes in cynomolgus macaques results in notable alterations in retinal thickness, particularly affecting the nasal inner retina. The observed increase in choriocapillaris thickness in both diabetic macaques and human patients with NPDR likely indicates pathologic changes and remodeling due to diabetes. Cynomolgus macaque presents a valuable NHP model for studying diabetic retinopathy.

Gangliosides and Cholesterol: Dual Regulators of Neuronal Membrane Framework in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with heterogeneous clinical presentation. Diagnosing ASD is complex, and the criteria for diagnosis, as well as the term ASD, have changed during the last decades. Diagnosis is made based on observation and accomplishment of specific diagnostic criteria, while a particular biomarker of ASD does not yet exist. However, studies universally report a disequilibrium in membrane lipid content, pointing to a unique neurolipid signature of ASD. This review sheds light on the possible role of cholesterol and gangliosides, complex membrane glycosphingolipids, in the development of ASD. In addition to maintaining membrane integrity, neuronal signaling, and synaptic plasticity, these lipids play a role in neurotransmitter release and calcium signaling. Evidence linking ASD to lipidome changes includes low cholesterol levels, unusual ganglioside levels, and unique metabolic profiles. ASD symptoms may be mitigated with therapeutic interventions targeting the lipid composition of membranes. However, restoring membrane equilibrium in the central nervous system remains a challenge. This review underscores the need for comprehensive research into lipid metabolism to uncover practical insights into ASD etiology and treatment as lipidomics emerges as a major area in ASD research.

ANALYSIS OF HENLE FIBER LAYER AND OUTER RETINAL LAYERS IN CONE DYSTROPHY.

To evaluate Henle fiber layer (HFL) thickness and volume parameters in patients with cone photoreceptor atrophy with directional optical coherence tomography. Macular 20°×20° standard and directional optical coherence tomography images were acquired from patients diagnosed with hereditary cone dystrophy with evident foveal ellipsoid zone defect in optical coherence tomography and age-matched healthy controls. Thickness and volume parameters of HFL, outer nuclear layer (ONL), and retinal layers between ellipsoid zone and Bruch membrane complex (EZ-BM) were calculated from manual segmentation through directional optical coherence tomography images, and comparative analysis is performed. Twelve eyes of six patients were compared with 12 eyes of six age-matched healthy controls (mean age: 29.5 ± 16.6 and 26.6 ± 3.9 years, respectively; P = 0.162). Patients had lower total HFL volume (0.45 ± 0.03 and 0.85 ± 0.15 mm 3 ; P < 0.001) and mean HFL thickness (16.1 ± 1.1 and 30.1 ± 5.3 µ m; P < 0.001) than healthy controls. Central subfield, parafoveal, and perifoveal ETDRS zone HFL parameters in patients were significantly lower than healthy controls. A centrifugal correlation was found between central outer nuclear layer and the corresponding parafoveal HFL (Spearman rho: 0.785; P < 0.001). Henle fiber layer assessment might be a useful optical coherence tomography biomarker in patients with cone photoreceptor atrophy. Henle fiber layer thinning is observed in foveal, parafoveal, and perifoveal areas of patients with cone photoreceptor atrophy, while volume reduction in outer nuclear layer and ellipsoid zone and Bruch membrane complex components were limited to central and parafoveal zones.

Lipid Curvature and Fluidity Influence Lipid Incorporation Disparities in Nanodiscs.

Nanodiscs have become a popular membrane mimetic system offering a well-defined bilayer environment to stabilize membrane proteins for in vitro analyses using a range of analytical methods; however, lipid compositions common to their deployment are simplistic and often fail to model native membrane complexity. Furthermore, there has been a general lack of rigorous analytical and biophysical characterization of nanodiscs comprising more than one lipid. To address these challenges, we coupled a nanodisc formation and purification workflow with targeted LC-MS/MS analysis to quantify lipids in nanodiscs made with different compositions. We screened lipids with a variety of headgroups and acyl chains and found that lipids did not always incorporate into nanodiscs at expected levels. Disparities in lipid incorporation were found to increase upon the addition of lipids known to induce curvature or rigidity to the membrane. Additionally, we found that adding just one additional type of lipid to nanodiscs changes the particle diameter and dispersity compared to nanodiscs containing a single lipid. We also formed and characterized nanodiscs using a complex starting composition inspired by the endoplasmic reticulum membrane and observed native-like cholesterol dynamics that modulated the lipid fluidity in the model bilayer system. Taken together, this work serves as a foundation for understanding nonstoichiometric lipid incorporation into nanodiscs and provides a basis for more thorough nanodisc characterization and quality control, which is critical to ensure multilipid nanodiscs synthesized accurately model the biological system of interest, enabling robust characterization of how the lipid landscape affects membrane protein structure and activity.

Two Variants of the ANK1 Gene Associated with Hereditary Spherocytosis

Background Hereditary spherocytosis (HS) is an erythrocytic membranopathy that belongs to a group of rare genetic disorders. Mutations in five genes, including ANK1, cause clinical manifestations of the disease. Identified variations in individual families provide a better understanding of the molecular basis of the disease. Methods In this study, we used two sequencing methods, whole exome sequencing (WES) and Sanger sequencing, analyzing gDNA and cDNA as templates, to detect and verify the variants putatively responsible for the clinical symptoms observed in a Polish family diagnosed with HS. Results We detected two variants that occur in cis in the ANK1 gene, a known missense mutation (NP_000028.3:p.V463I) and a novel frameshift mutation (NP_000028.3: p.V1626fs*64) that appears to be crucial for the probands. As shown by transcriptome studies, the mutant allele is not present at a detectable level. Conclusions We conclude that the molecular basis of this case is related to an unstable transcript of the mutant allele and that the direct cause of the HS is a deficiency of erythrocyte ankyrin leading to a disruption of the AE1-erythrocyte ankyrin-spectrin complex in the erythrocyte membrane.

Enhancement of Oryzanol application by constructing modified β-CD inclusion complex and polycaprolactone-chitosan electrospun fiber membranes: Perspectives on wound dressings and grape preservation.

Oryzanol has a variety of physiological activities and is widely used in food and medicine. However, its utilization form and bioavailability are limited by poor solubility and photothermal stability. In this paper, an inclusion complex (IC) was prepared by modifying β-cyclodextrin as a molecular carrier to encapsulate Oryzanol. Polycaprolactone-chitosan (PCL-CS) and IC were prepared into a fiber membrane (PCL-CS-IC) using an electrostatic spinning technique and applied to wound healing and grape preservation. The results showed that the prepared ICs had a drug loading rate of 43.18% with good antimicrobial, wettability, and air permeability properties. The PCL-CS-IC effectively reduced the inflammation of mouse wounds, with obvious re-epithelialization and inflammatory factors reduction in skin tissues. Meanwhile, the PCL-CS-IC delayed the decay process of grapes and extended shelf life. In conclusion, this study effectively improved the utilization of oryzanol and provided potential ideas for wound dressing preparation and food packaging materials development.

Inhibition of the gut sulfidogenic bacteria targeting the production of toxic and inflammatory sulfide

Abstract Background and aims This project seeks to develop novel strategies to modulate the human gut microbiota and mitigate the production of pathogenic sulphide strongly associated with inflammatory bowel diseases (IBD). Our focus lies in inhibiting a key respiratory complex present in majority of the gut sulfidogenic bacteria to curb excessive sulphide production. Methods We will use an array of genetic, microbiological, and biochemical techniques to perform both in vitro and in vivo experiments. Our primary target is the membrane complex DsrMKJOP, involved in the essential Dsr pathway in sulfidogenic bacteria, which is pivotal for sulphur respiration. To achieve this, we will evaluate the efficacy of inhibitors, specifically quinone analogues, which compete for binding sites in the DsrMKJOP complex. Our comprehensive work plan comprises two main components: (1) expression and production of the DsrMKJOP complex from a major sulfidogenic bacterium and investigation of its inhibition profile through in vitro enzymatic assays; and (2) assessment of the efficacy and toxicity of DsrMKJOP inhibitors in vivo. This includes determining minimal inhibitory and bactericidal concentrations for the two bacteria, evaluating cytotoxicity in human colonic cells, assessing toxicity in a non-vertebrate animal model, and determining the antimicrobial properties of the inhibitory drugs to modulate bacterial growth during infection and colonization. Anticipated impact The outcomes of this research will contribute to identifying new therapeutic agents against sulfidogenic bacteria, acting as metabolic modulators to inhibit the production of pathogenic inflammatory sulphide. We aim to not only identify novel DsrMKJOP inhibitors but also to characterize their in vivo efficacy and toxicity profiles. Successful outcomes hold the potential to significantly advance human health, offering promising avenues for preventing IBD, enhancing the quality of life for IBD patients, and alleviating the burden on healthcare systems.

Sulfated Glycosaminoglycans as Inhibitors for Chlamydia Infections: Molecular Weight and Sulfation Dependence.

Glycosaminoglycans (GAGs) play a pivotal role in pathogen attachment and entry into host cells, where the interaction with GAGs is critical for a diverse range of bacteria and viruses. This study focuses on elucidating the specific interactions between sulfated GAGs and the adhesin OmcB (Outer membrane complex protein B) of Chlamydia species, examining how structural characteristics of GAGs, such as sulfation degree and molecular weight, influence their binding affinity and thereby affect bacterial infectivity. A surface-based binding assay is established to determine the binding constants of OmcB with various GAGs. It is shown that increased sulfation and higher molecular weight enhance GAG binding to OmcB. These findings are further validated using cell assays, which shows that the addition of sulfated GAGs reduces OmcB-cell binding and inhibits the attachment of C. pneumoniae elementary bodies (EBs), underscoring the pivotal role of specific GAGs in chlamydial infections. Notably, heparin exhibites a stronger inhibitory effect on OmcB compare to GAGs with similar sulfation degrees and molecular weights, suggesting that particular molecular architectures may optimize binding interactions.

Protective Efficacy of Decreasing Antigen Doses of a Chlamydia abortus Subcellular Vaccine Against Ovine Enzootic Abortion in a Pregnant Sheep Challenge Model.

Chlamydia abortus, the cause of ovine enzootic abortion, is a zoonotic bacterial pathogen and one of the most infectious causes of foetal death in sheep worldwide. Although the disease can be controlled using commercial inactivated and live whole-organism vaccines, there are issues with both, particularly concerning efficacy and safety. Recently, we have described the development of a new COMC (chlamydial outer membrane complex) vaccine based on a detergent-extracted outer membrane protein preparation of the pathogen, which can be delivered in a single inoculation and is both efficacious and safe. In this study, we have evaluated the COMC vaccine further in a dose-response titration of the chlamydial antigen content of the vaccine (from 20 to 2.5 µg in seven experimental groups) using an established pregnant sheep challenge model. No obvious dose-response relationship was observed across the groups, with a single abortion event occurring in four of the groups and three in the lowest dose group (2.5 µg). No abortions occurred in the 15 and 10 µg groups. The abortion rates (0-14%) were significantly below that of the challenge control group (33%). A similar reduction in bacterial shedding of infectious organisms following parturition was observed in the vaccinated groups compared to the challenge control group, which is important in terms of reducing potential transmission to naive animals. The results show that a dose of 10 µg antigen in the vaccine will be optimal in terms of maximising efficacy, reducing shedding at parturition, and ensuring it is cost-effective to produce for commercial manufacture.

Outcomes of intravitreal dexamethasone implant (Ozurdex®) in patients with post-surgical macular edema – a real-world scenario

BackgroundClinically significant macular edema (CME) is the leading cause of visual loss after ophthalmologic surgery due to the release of inflammatory mediators promoted by the procedures. We aimed to evaluate the outcomes of intravitreal Ozurdex® (700 µg dexamethasone) implants as a primary therapeutical option for post-surgical macular edema cases.MethodsPatients with post-surgical macular edema diagnosed by optical coherence tomography (Cirrus SD-OCT) and treated with Ozudex were selected for the current study. Data was retrospectively collected from medical records from January 2020 to December 2022 and included sex, age, laterality, and timeline of treatment (i.e. implant alone or at the time of silicon oil removal in cases requiring vitreorretinal surgery). Complications associated with treatment were also noted as well as the need of further treatments. The structural analysis focused on measuring central macular thickness (CMT—average thickness within the 1 mm circle of the ETDRS) from the internal limiting membrane to the Bruch’s membrane complex, as well as the average total macular thickness including parafoveal and perifoveal regions determined by the device (CAT). The functional evaluation was based on the best-corrected visual acuity (VA) measured in logMAR.ResultsA total of 46 participants were included (56.2% males, mean age: 60.9 ± 11.2 years old). A statistically significant change was observed in the postoperative versus the preoperative period for all parameters (p < 0.05). The mean VA difference was − 0.17 ± 0.24; CMT was − 109.22 ± 124.85 and CAT was − 14.76 ± 58.95. We observed a significant effect of the moment of Ozurdex implantation on VA improvement, so that cases with implantation at the time of oil removal showed lower improvement than cases with implantation at a distinct timing (Coef. 0.19, 95%CI: 0.02 to 0.36, p = 0.027). Eleven cases (23.9%) required further treatment such as new Ozurdex implantation (8 cases) or surgery (3 cases). Only one case (2.17%) showed increased intraocular pressure and underwent glaucoma surgery.ConclusionsIntravitreal Ozurdex implants significantly improved functional and structural aspects in post-surgical macular edema. The timing of implantation influenced VA improvement, with a distinct step approach showing better outcomes than at the time of oil removal.

Adaptive sampling-based structural prediction reveals opening of a GABAA receptor through the αβ interface.

γ-Aminobutyric acid type A (GABAA) receptors are ligand-gated ion channels in the central nervous system with largely inhibitory function. Despite being a target for drugs including general anesthetics and benzodiazepines, experimental structures have yet to capture an open state of classical synaptic α1β2γ2 GABAA receptors. Here, we use a goal-oriented adaptive sampling strategy in molecular dynamics simulations followed by Markov state modeling to capture an energetically stable putative open state of the receptor. The model conducts chloride ions with comparable conductance as in electrophysiology measurements. Relative to experimental structures, our open model is relatively expanded at both the cytoplasmic (-2') and central (9') gates, coordinated with distinctive rearrangements at the transmembrane αβ subunit interface. Consistent with previous experiments, targeted substitutions disrupting interactions at this interface slowed the open-to-desensitized transition rate. This work demonstrates the capacity of advanced simulation techniques to investigate a computationally and experimentally plausible functionally critical of a complex membrane protein yet to be resolved by experimental methods.

Regulation of mutant huntingtin mitochondrial toxicity by phosphomimetic mutations within its N-terminal region.

Huntington's disease (HD), a neurodegenerative disease, affects approximately 30,000 people in the United States, with 200,000 more at risk. Mitochondrial dysfunction caused by mutant huntingtin (mHTT) drives early HD pathophysiology. mHTT binds the translocase of mitochondrial inner membrane (TIM23) complex, inhibiting mitochondrial protein import and altering the mitochondrial proteome. The HTT N-terminal 17-amino acids sequence (N17) acts as a regulatory domain in HD pathogenesis; phosphomimetic modification of serine 13 and 16 of the N17 domain impacts subcellular localization, degradation, and ameliorates toxicity in mouse and cell models of HD. Using cellular and mouse (either sex) HD models, we investigated the mechanisms by which HTT phosphorylation affects intracellular localization. We demonstrate that introducing phosphomimetic mutations within the mHTT fragment N17 domain decreased TIM23 binding affinity and reduced inhibition of mHTT-mediated mitochondrial protein import. BACHD-SD mice expressing full-length mHTT harboring the same two N17 phosphomimetic mutations have an ameliorated HD-like phenotype as compared to mice expressing mHTT. Consistent with reduced toxicity in vivo, we found that the amount of full-length mHTT in brain mitochondria of BACHD-SD transgenic mice is less when the mHTT has two phosphomimetic mutations. To complement the relevance of the phosphomimic HTT findings, endogenous N17 phospho mHTT is less likely to translocate to the mitochondria compared to non-phosphorylated mHTT. We demonstrate that phosphorylation of mHTT at serines 13 and 16 is critical for negatively regulating mHTT mitochondrial targeting and that reducing mHTT mitochondrial localization and binding to TIM23 results in amelioration of mHTT-induced mitochondrial and neuronal toxicity.Significance Statement We establish the first 17 amino acids of mHTT as a mitochondrial targeting sequence driving mHTT accumulation in mitochondria and show that N-terminal phosphomimetic modifications reduce mitochondrial accumulation and toxicity. Utilizing super-resolution live imaging and expansion microscopy of isolated mitochondria and full-length mHTT immunoprecipitation from human cells, we demonstrate HTT intramitochondrial localization and interaction with the mitochondrial protein importing complex. mHTT N17 domain phosphomimetic mutations reduce binding affinity for the TIM23 subunit, preventing mHTT mitochondrial accumulation. This lessens mHTT inhibitory effect on mitochondrial protein import. Our study elucidates the molecular mechanism responsible for the BACHD-SD mouse ameliorated phenotype and underscores a critical need to identify enzymes responsible for regulating HTT phosphorylation to exploit their potential as therapeutic HD targets.

Mitochondrial YME1L1 governs unoccupied protein translocase channels.

Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase fused to the N terminus of the inner membrane protein MIC60. While stabilization of the dihydrofolate reductase domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, notably, plugging the channel of translocase of the outer membrane) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 seems to play a crucial role in mitochondrial quality control to counteract precursor stalling in the translocase of the outer membrane complex and unoccupied TIM23 channels.

Reacting to reductive stress at the mitochondrial import gate.

A byproduct of mitochondrial energy production is the generation of reactive oxygen species (ROS). Too much ROS is toxic, but ROS deficiency is equally deleterious (reductive stress). In a recent study, McMinimy et al. uncovered a ubiquitin proteasome-mediated mechanism at the translocase of the outer membrane (TOM) complex, which senses ROS depletion and adjusts mitochondrial protein import accordingly.

Fluorescent Probes to Track Complex Membrane Blebbing

Fluorescent Probes to Track Complex Membrane Blebbing

Phagocytosis by the retinal pigment epithelium: New insights into polarized cell mechanics.

The retinal pigment epithelium (RPE) is a specialized epithelium at the back of the eye that carries out a variety of functions essential for visual health. Recent studies have advanced our molecular understanding of one of the major functions of the RPE; phagocytosis of spent photoreceptor outer segments (POS). Notably, a mechanical link, formed between apical integrins bound to extracellular POS and the intracellular actomyosin cytoskeleton, is proposed to drive the internalization of POS. The process may involve a "nibbling" action, as an initial step, to sever outer segment tips. These insights have led us to hypothesize an "integrin adhesome-like" network, atypically assembled at apical membrane RPE-POS contacts. I propose that this hypothetical network orchestrates the complex membrane remodeling events required for particle internalization. Therefore, its analysis and characterization will likely lead to a more comprehensive understanding of the molecular mechanisms that control POS phagocytosis.

Role of the CaV1.2 distal carboxy terminus in the regulation of L-type current

ABSTRACT L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVβ2b. The CaV1.2 channel’s C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels’ basal activity in cardiomyocytes.

Cascading expression of ApiAP2 transcription factors controls daughter cell assembly in Toxoplasma gondii.

Pathogenesis of Toxoplasma gondii in the intermediate host is based on the tachyzoite ability to divide rapidly to produce significant amount of daughter cells in a reduce time frame. The regulation of the cell-cycle specific expression program is therefore key to their proliferation. Transcriptional regulation has a crucial role in establishing this expression program and transcription factors regulates many aspects of tachyzoite cell cycle. We explored the role of two ApiAP2 transcription factors, TgAP2XII-9 and TgAP2III-2, during the cell cycle of the tachyzoite form. While TgAP2III-2 has only a minor impact on the tachyzoite proliferation, we show that TgAP2XII-9 regulates many aspects of the cell cycle including the proper assembly of the daughter cells inner membrane complex and temporal expression of many virulence genes. Creation of a double mutant strain for TgAP2XII-9 and TgAP2III-2 shows that TgAP2XII-9 had a prominent role during daughter cell assembly. Using transcriptomics and Cut&Tag, we demonstrate that TgAP2XII-9 mainly acts through the transcriptional control of at least 300 genes promoters. Interestingly, TgAP2XII-9 plays a crucial role repressing the expression of genes necessary for budding initiation and activating genes necessary for microneme de novo formation. We also explored the importance of the AP2 domain of TgAP2XII-9 demonstrating its critical role to exert its function. Therefore, we showed that TgAP2XII-9 is a crucial transcription factor which is key to daughter cell assembly post budding initiation.

Aqueous Synthesis of Membrane Lipids via Amide Formation between Amphiphilic Amines and Thioacids

Protocell membranes were likely formed through primitive metabolic reactions that synthesized more complex membrane lipid species from simpler precursors. Thioacids have been shown to be prebiotically plausible and chemoselective N‐acylating reagents. They have been successfully employed for the acylation of peptides and RNA. However, the ability of thioacids to acylate lipid species has not yet been explored. Here, we demonstrate N‐acylation of natural amine‐containing amphiphiles using fatty thioacids. This reaction occurs spontaneously in aqueous conditions, is chemoselective, and traceless. The synthesis of several natural sphingolipids via direct N‐acylation is possible and their subsequent de novo formation into vesicular membranes is shown. Aqueous thioacid coupling provides a route for generating membrane‐forming lipids in the context of protocells and for the in situ synthesis of relevant sphingolipid species.

Endothelial Response to Blood-Brain Barrier Disruption in the Human Brain.

Cerebral endothelial cell (EC) injury and blood-brain barrier (BBB) permeability contribute to neuronal injury in acute neurological disease states. Preclinical experiments have used animal models to study this phenomenon, yet the response of human cerebral ECs to BBB disruption remains unclear. In our Phase 1 clinical trial (NCT04528680), we used low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce transient BBB disruption of peri-tumoral brain in patients with recurrent glioblastoma. We found radiographic evidence that BBB integrity was mostly restored within 1-hour of this procedure. Using single-cell RNA sequencing and transmission electron microscopy, we analyzed the acute response of human brain ECs to ultrasound-mediated BBB disruption. Our analysis revealed distinct EC gene expression changes after LIPU/MB, particularly in genes related to neurovascular barrier function and structure, including changes to genes involved in the basement membrane, EC cytoskeleton, and junction complexes, as well as caveolar transcytosis and various solute transporters. Ultrastructural analysis showed that LIPU/MB led to a decrease in luminal caveolae, the emergence of cytoplasmic vacuoles, and the disruption of the basement membrane and tight junctions, among other things. These findings suggested that acute BBB disruption by LIPU/MB led to specific transcriptional and ultrastructural changes and could represent a conserved mechanism of BBB repair after neurovascular injury in humans.