Fluids and Barriers of the CNS

BackgroundThe recent characterization of the glymphatic system and meningeal lymphatics has re-emphasized the role of the meninges in facilitating CSF transport and clearance. Here, we characterize small and large CSF solute distribution patterns along the intracranial and surface meninges in neonatal rodents and compare our findings to a rodent model of intraventricular hemorrhage-posthemorrhagic hydrocephalus. We also examine CSF interactions with the tela choroidea and its pial invaginations into the choroid plexuses of the lateral, third, and fourth ventricles. Methods1.9-nm gold nanoparticles, 15-nm gold nanoparticles, or 3 kDa Red Dextran Tetramethylrhodamine constituted in aCSF were infused into the right lateral ventricle of P7 rats to track CSF circulation. 10 minutes post-1.9-nm gold nanoparticle and Red Dextran Tetramethylrhodamine injection and 4 hours post-15-nm gold nanoparticle injection, animals were sacrificed and brains harvested for histologic analysis to identify CSF tracer localization in the cranial and spine meninges and choroid plexus. Spinal dura and leptomeninges (arachnoid and pia) wholemounts were also performed. ResultsThere was significantly less CSF tracer distribution in the dura compared to the arachnoid and pia maters in neonatal rodents. Both small and large CSF tracers were transported intracranially to the arachnoid and pia mater of the perimesencephalic cisterns and tela choroidea, but not the dura mater of the falx cerebri. CSF tracers followed a similar distribution pattern in the spinal meninges. In the choroid plexus, there was large CSF tracer distribution in the apical surface of epithelial cells, and small CSF tracer along the basolateral surface. There were no significant differences in tracer intensity in the intracranial meninges of control vs. intraventricular hemorrhage-posthemorrhagic hydrocephalus (PHH) rodents, indicating preserved meningeal transport in the setting of PHH. ConclusionsDifferential CSF tracer handling by the leptomeninges suggests that there are distinct roles for CSF handling between the arachnoid-pia and dura maters in the developing brain. Similarly, differences in apical vs. luminal choroid plexus CSF handling may provide insight into particle-size dependent CSF transport at the CSF-choroid plexus border.

The blood-tumor barrier (BTB) prevents effective central nervous system (CNS) drug delivery, especially in malignant gliomas. Brain endothelium predominates the BTB and connects through bicellular and tricellular tight junctions (TJ). Angulin-1/LSR, is a highly expressed endothelial tricellular TJ. Our studies explore the role of Angubindin-1, an Angulin-1/LSR binder, to disrupt tricellular TJ integrity, increase drug entry and hamper glioma progression. Using rat brain endothelial cells (RBMVEC) we tracked Angulin-1/LSR localization and expression to the membrane; binding tightest to Angubindin-1 2-8 hours post-treatment (p < 0.05). Angubindin-1 dose-dependently reduced bicellular and tricellular TJs 1-4 hours post treatment (p < 0.05), returning to baseline by 24 hours (p < 0.05). In human and rat-derived glioma cells, Angubindin-1 transiently reduced Angulin-1/LSR expression between 2-8 hours (p < 0.05), with return to baseline by 24 hours (p < 0.001). Silenced Angulin-1/LSR expression on endothelium resulted in decreased mRNA levels of bicellular (occludin, claudin-5, ZO-1) and tricellular (tricellulin/MARVELD2, angulin-1/LSR) TJs compared to control (p < 0.01). Angubindin-1 treatment also inhibited efflux transporter P-gp in both RBMVECs and glioma cells with high P-gp expression only. Orthotopic rat glioma models were treated with Doxil (3 mg/kg), Angubindin-1 (10 mg/kg), or combination to evaluate BTB permeability/drug accumulation, and overall survival. Combination therapy enhanced Doxil tumor accumulation by 20% (p < 0.001), reduced tumor volume by day 14 (77.5% vs. 81.6%, p < 0.05), and significantly extended survival compared to Doxil alone (24 days vs. 18 days, p < 0.0001). These findings demonstrate the effects of tricellular tight junction inhibition on disrupting the BTB, enhancing CNS drug delivery, and improving rodent glioma survival. SignificanceThis study demonstrates that Angubindin-1, a targeted modulator of tricellular tight junction protein Angulin-1/LSR, transiently disrupts BTB integrity to enhance chemotherapy delivery and prolong survival in glioma-bearing rats. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/667901v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@b1b46corg.highwire.dtl.DTLVardef@bc552dorg.highwire.dtl.DTLVardef@7c1a74org.highwire.dtl.DTLVardef@1ace101_HPS_FORMAT_FIGEXP M_FIG C_FIG Angubindin-1 targets both bicellular tight junctions and the tricellular tight junction protein, Angulin-1/LSR, in brain endothelial and glioma cells leading to transient disruption of the blood-tumor barrier (BTB) and inhibition of P-glycoprotein towards enhanced Doxil penetration and reduced tumor burden.

We have established and optimized a protocol for the high-yield isolation of primary epithelial cells from rat choroid plexus. The addition of cytosine arabinoside suppressed the growth of contaminating cells, and epithelial culture was grown into a confluent impermeable monolayer within 5-6 days after seeding. To form an in vitro blood-CSF barrier, epithelial cells were plated on inverted coated polycarbonate support of Transwell inserts. Morphologically, the polarized cells remained cuboidal in shape and expressed TJ proteins at a high rate. The filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 160 to 180 {Omega} x cm2 and remained at this level for 3 days, indicating the persistent formation of continuous TJs. The cells were able to secrete cerebrospinal fluid (CSF) actively. Epithelial cells showed expression of selective influx and efflux transporters. To conclude, our BCSFB model exhibits tight, functional barrier characteristics and shows the functional expression of the pharmaceutically important influx/efflux transporters. The recent model is suitable for in vitro investigations of BCSFB and routine pre-clinical drug discovery.

This study investigates the impact of rapamycin and propranolol on cerebral cavernous malformations (CCMs). Employing an unbiased transcriptomic analysis, we aimed to comprehensively elucidate the molecular mechanisms underlying these drug effects in Mouse Brain Microvascular Endothelial Cells (mBMEC) deficient in Ccm3. While propranolol shows limited efficacy in modulating the CCM transcriptomic phenotype in mBMEC, rapamycin demonstrates a higher impact. Rapamycin reverses gene expression changes induced by Ccm3 deficiency, restoring Klf2/4-dependent genes like Nos3, Adamts1, and Thbs1. Notably, we observed a reduction in KLF2 protein levels in Ccm3 KO cells treated with rapamycin. Critically, in vivo experiments demonstrate that a combination of rapamycin and lapatinib effectively reduces lesion volume in a chronic CCM model. This finding is particularly noteworthy as it suggests a potential treatment strategy for existing lesions. In summary, our work describes a new mechanism for the effects of rapamycin in Ccm3- deficient cells and identifies a new drug combination in the treatment of cavernomas.

Cerebral Cavernous Malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system (CNS) upon loss of the CCM multimeric protein complex. The endothelial cells (ECs) within CCM lesions are characterised by overactive MEKK3 kinase and KLF2/4 transcription factor signalling, leading to pathological changes such as increased EC spreading and reduced junctional integrity. Concomitant to aberrant EC signalling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the CNS. Here, we adapted a three dimensional (3D) microfluidic system to examine CCM1 deficient human micro-vessels in distinctive ECMs. We validate that EC pathological hallmarks are maintained in this 3D model. We further show that key genes responsible for homeostasis of Hyaluronic Acid (HA), a major ECM component of the CNS, are dysregulated in CCM. Supplementing the ECM in our model with forms of HA that are predicted to be reduced, inhibits CCM cellular phenotypes, independent of KLF2/4. This study thereby provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signalling impact vascular malformations.

BackgroundInside the incompressible cranium, the volume of cerebrospinal fluid (CSF) is directly linked to blood volume: a change in either will induce a compensatory change in the other. Vasodilatory lowering of blood pressure has been shown to result in an increase of intracranial pressure, which, in normal circumstances should return to equilibrium by increased fluid efflux. In this study, we investigated the effect of blood pressure lowering (BPL) on fluorescent CSF tracer absorption into the systemic blood circulation. MethodsBPL was performed by an i.v. administration of nitric oxide donor sodium nitroprusside (5 {micro}g kg-1 min-1) or the Ca2+-channel blocker nicardipine hydrochloride (0.5 {micro}g kg-1 min-1) for 10 and 15 to 40 mins, respectively. The effect of BPL on CSF clearance was investigated by measuring the efflux of fluorescent tracers (40 kDa FITC-dextran, 45 kDa Texas Red-conjugated ovalbumin) into blood and deep cervical lymph nodes. ResultsNicardipine and sodium nitroprusside reduced blood pressure by 32.0 {+/-} 19.6% and 22.0 {+/-} 2.5%, while temporarily elevating in intracranial pressure by 14.0 {+/-} 6.0% and 11.6 {+/-} 2.0%, respectively. BPL significantly increased tracer accumulation into deep cervical lymph nodes and systemic circulation, but reduced perivascular inflow along penetrating arteries in the brain. The enhanced tracer efflux by BPL into the systemic circulation was markedly reduced (-66.7%) by ligation of lymphatic vessels draining into deep cervical lymph nodes. ConclusionsThis is the first study showing that CSF clearance can be improved with acute hypotensive treatment and that the effect of the treatment is reduced by ligation of a lymphatic drainage pathway. Enhanced CSF clearance by BPL may have therapeutic potential in diseases with dysregulated CSF flow.

Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we employed in vivo and ex vivo approaches to reveal the efficacy and mode of action of AZE in the rat brain. The drug effectively reduced the ICP, irrespective of the mode of drug administration and level of anaesthesia. The effect occurred via a direct action on the choroid plexus and an associated decrease in cerebrospinal fluid secretion, and not indirectly via the systemic action of AZE on renal and vascular processes. Upon a single administration, the reduced ICP endured for approximately 10 h post-AZE delivery with no long-term changes of brain water content or choroidal transporter expression. However, a persistent reduction of ICP was secured with repeated AZE administrations throughout the day. Future specific targeting of choroidal carbonic anhydrases may limit the systemic side effects, and therefore enhance the treatment tolerability and effectiveness in select patient groups experiencing elevated ICP.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

The blood-brain barrier (BBB) is central to separate blood from the extracellular fluids of the brain. To understand disease-related changes in the BBB is pivotal and such changes can increasingly be studied by single-cell RNA sequencing (scRNAseq), which provides high-resolution insight into gene expression changes related to the pathophysiological response of the vasculature. However, analysis of the vascular cells in the brain is challenging due to the low abundance of these cells relative to neuronal and glial cells, and improved techniques for enrichment of the vascular component is therefore warranted. The present study describes a method whereby panning with CD31-coated magnetic beads allows isolation of brain vasculature without the need for transgenic reporter lines or FACS sorting. The protocol was tested in three modalities: isolation of cells for scRNAseq, western blot (WB) analysis, and primary cell culture. For scRNAseq, a total of 22,515 single-cell transcriptomes were generated from 12-months old mice and separated into 23 clusters corresponding to all known vascular and perivascular cell types. The most abundant cell type was endothelial cells (EC) (Pecam1- and Cdh5-positive), which dispersed into clusters of arterial, capillary, and venous EC according to previously established BBB arterio-venous zonation markers. Furthermore, we identified clusters of microglia (Aif1-positive), one cluster of fenestrated endothelial cells (Plvap-positive; Cldn5-negative), a cluster of pericytes (Kcnj8- and Abcc9-positive) and a cluster of vascular smooth muscle cells (VSMC) (Acta2- and Tagln-positive). WB analysis using established markers for the different cell types (CD31 (EC), SM22 (VSMC), PDGFRB (pericytes), GFAP (astrocytes), and IBA1 (microglia) confirmed their presence in the isolated vascular component and suggests that the protocol is suitable for future proteomic analysis. Finally, we adapted the isolation protocol to accommodate primary culture of brain vascular cells. In conclusion, we have successfully established a simple and fast method for isolating microvasculature from the murine brain independent of cell sorting and alleviating the need to use reporter mouse lines. The protocol is suitable for a multitude of testing modalities, including single-cell analyses, WB and primary cell culture.

AO_SCPLOWBSTRACTC_SCPLOWSorCS2 is involved in trafficking of membrane receptors and transporters. SorCS2 is implicated in brain disorders, but the mechanism remains uncertain. We hypothesized that SorCS2 expression is important for neurovascular coupling. Brains from P8 and 2-month-old wild type mice were stained for SorCS2 and compared to SorCS2 knockouts (Sorcs2-/-). Changes in cerebral perfusion in response to sensory stimulation, i.e., neurovascular coupling, were compared in vivo. Neurovascular coupling was also assessed ex vivo in brain slices loaded with calcium-sensitive dye. Proteomics of astrocytes was analyzed for ingenuity pathways. SorCS2 was strongly expressed in astrocytic endfeet of P8 mice but only in few astrocytes from 2-month-old brains. Sorcs2-/- mice demonstrated reduced neurovascular coupling. This was associated with reduced astrocytic calcium response to neuronal excitation in Sorcs2-/- mice. No difference in cerebral artery caliber nor in endothelial function was seen between wild type and Sorcs2-/- mice. Proteomics indicated reduced glutamatergic signaling and suppressed calcium signaling in Sorcs2-/- astrocytes. We suggest that SorCS2 expression is important for neurovascular coupling due to modulation of glutamatergic and calcium signaling in astrocytes.

Cerebral cavernous malformations (CCM), also known as cavernous angiomas, are blood vessel abnormalities comprised of clusters of grossly enlarged and hemorrhage-prone capillaries. The prevalence in the general population, including asymptomatic cases, is estimated to be 0.5%. Some patients develop severe symptoms, including seizures and focal neurologic deficits, while others have no symptoms. The causes of this remarkable presentation heterogeneity within a primarily monogenic disease remain poorly understood. To address this problem, we have established a chronic mouse model of CCM, induced by postnatal ablation of Krit1 with Pdgfb-CreERT. These mice develop CCM lesions gradually over 4-6 months of age throughout of the brain. We examined lesion progression in these mice with T2-weighted 7T MRI protocols. Precise volumetric analysis of individual lesions revealed non-monotonous behavior, with some lesions temporarily growing smaller. However, the cumulative lesional volume invariably increased over time and accelerated after about 3 months. Next, we established a modified protocol for dynamic contrast enhanced (DCE) MR imaging and produced quantitative maps of gadolinium tracer MultiHance in the lesions, indicating a high degree of heterogeneity in lesional permeability. Multivariate comparisons of MRI properties of the lesions with cellular markers for endothelial cells, astrocytes, and microglia revealed that increased cell density surrounding lesions correlates with stability, while increased vasculature within and surrounding lesions may correlate with instability. Our results lay a foundation for better understanding individual lesion properties and provide a comprehensive pre-clinical platform for testing new drug and gene therapies for controlling CCM.

BackgroundSurgical insertion of a ventricular shunt initiates a cytokine response shown to play a role in shunt failure caused by obstruction. These pro-inflammatory and anti-inflammatory cytokines cause astrocytes, amongst others, to enter an activated state which causes an increase in attachment. 4,5-Dihydro-3-phenyl-5-isoxazoleacetic acid (GIT 27) is a reagent with immunomodulatory properties which acts by blocking the main signaling protein on astrocytes and microglia called toll-like receptor 4 (TLR-4). MethodsIn this experiment, we tested the effect of GIT 27 on astrocytes when used as a pre-treatment, simultaneous treatment, and post-treatment relative to shunt insertion represented by the introduction of IL-1{beta} or IL-10. Control, DMSO vehicle control, and GIT 27 treated sample groups were assayed for cell counts and cytokine concentration data. ResultsExposure of astrocytes to suspended GIT 27 in a DMSO vehicle caused a decrease in cell attachment and a significant decrease in the concentration of the majority of cytokines. Comparisons of GIT 27 exposure times, represented by pre-, simultaneous, and post-treatment groups, showed that pre-treatment with GIT 27 is most effective at decreasing cellular attachment where post-treatment was generally the most effective at decreasing pro-inflammatory cytokine concentrations. In future practice, this could be embodied by pharmacologic dosing prior to shunting and/or slow release from the shunt surface. ConclusionsGIT 27 is most effective at decreasing cell counts and cytokines when in-suspension compared to when attached to the shunt surface. Our data show that GIT 27 has the potential to be used as an effective way to modulate the cytokine response associated with shunt insertion.

BackgroundThe blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. MethodsA mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H -neurotensin, caused >8{degrees}C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M156,96,102His-13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H -13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1R56H, P96H, Y102H -13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

BackgroundGrowing evidence suggests that for rodents, a substantial fraction of cerebrospinal fluid (CSF) drains by crossing the cribriform plate into the nasopharengeal lymphatics, eventually reaching the cervical lymphatic vessels (CLVs). Disruption of this drainage pathway is associated with various neurological disorders. MethodsWe employ a lumped parameter method to numerically model CSF drainage across the cribriform plate to CLVs. Our model uses intracranial pressure as an inlet pressure and central venous blood pressure as an outlet pressure. The model incorporates initial lymphatic vessels (modeling those in the nasal region) that absorb the CSF and collecting lymphatic vessels (modeling CLVs) to transport the CSF against an adverse pressure gradient. To determine unknown parameters such as wall stiffness and valve properties, we utilize a Monte Carlo approach and validate our simulation against recent in vivo experimental measurements. ResultsOur parameter analysis reveals the physical characteristics of CLVs. Our results suggest that the stiffness of the vessel wall and the closing state of the valve are crucial for maintaining the vessel size and volume flow rate observed in vivo. We find that a decreased contraction amplitude and frequency leads to a reduction in volume flow rate, and we test the effects of varying the different pressures acting on the CLVs. Finally, we provide evidence that branching of initial lymphatic vessels may deviate from Murrays law to reduce sensitivity to elevated intracranial pressure. ConclusionsThis is the first numerical study of CSF drainage through CLVs. Our comprehensive parameter analysis offers guidance for future numerical modeling of CLVs. This study also provides a foundation for understanding physiology of CSF drainage, helping guide future experimental studies aimed at identifying causal mechanisms of reduction in CLV transport and potential therapeutic approaches to enhance flow.

Defects in brain endothelial cells (brainECs) can cause severe cerebrovascular malformations, including arteriovenous malformation (AVM) and cerebral cavernous malformation (CCM). The lack of appropriate tools for cerebrovascular disease modeling and local genetic manipulation of the brain vasculature hinders research on cerebrovascular malformations. Here we develop a recombinant adeno-associated virus (rAAV) tool miniBEND (rAAV-based mini-system for brain endothelial cells, rAAV-miniBEND), which combines a minimal promoter and an optimized cis-acting element (cis-element) isolated from the mouse gene Tek. This system achieves gene expression specifically in mouse and rat brainECs. Using rAAV-miniBEND, we achieve high-efficiency and high-specificity gene expression in brainECs through intracranial injection at various developmental stages and through intravenous administration at all postnatal stages in mice. Furthermore, we use rAAV-miniBEND to model sporadic CCMs mediated by MAP3K3I441M and AVMs mediated by BrafV600E. We demonstrate that somatic expression of BrafV600E in brainECs induces an AVM phenotype, and that brainEC proliferation are important for AVM development. Thus, our rAAV-miniBEND system provides a valuable and widely applicable tool for cerebrovascular disease modeling and local or global brainEC gene delivery.

Evidence strongly suggests that SARS-CoV-2, the cause of COVID-19, can enter the brain. SARS-CoV-2 enters cells via the S1 subunit of its spike protein, and S1 can be used as a proxy for the uptake patterns and mechanisms used by the whole virus; unlike studies based on productive infection, viral proteins can be used to precisely determine pharmacokinetics and biodistribution. Here, we found that radioiodinated S1 (I-S1) readily crossed the murine blood-brain barrier (BBB). I-S1 from two commercial sources crossed the BBB with unidirectional influx constants of 0.287 {+/-} 0.024 L/g-min and 0.294 {+/-} 0.032 L/g-min and was also taken up by lung, spleen, kidney, and liver. I-S1 was uniformly taken up by all regions of the brain and inflammation induced by lipopolysaccharide reduced uptake in the hippocampus and olfactory bulb. I-S1 crossed the BBB completely to enter the parenchymal brain space, with smaller amounts retained by brain endothelial cells and the luminal surface. Studies on the mechanisms of transport indicated that I-S1 crosses the BBB by the mechanism of adsorptive transcytosis and that the murine ACE2 receptor is involved in brain and lung uptake, but not that by kidney, liver, or spleen. I-S1 entered brain after intranasal administration at about 1/10th the amount found after intravenous administration and about 0.66% of the intranasal dose entered blood. ApoE isoform or sex did not affect whole brain uptake, but had variable effects on olfactory bulb, liver, spleen, and kidney uptakes. In summary, I-S1 readily crosses the murine BBB, entering all brain regions and the peripheral tissues studied, likely by the mechanism of adsorptive transcytosis. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Given the persistent challenge of differentiating idiopathic Normal Pressure Hydrocephalus (iNPH) from similar clinical entities, we conducted an in-depth proteomic study of cerebrospinal fluid (CSF) in 28 shunt-responsive iNPH patients, 38 Mild Cognitive Impairment (MCI) due to Alzheimers disease, and 49 healthy controls. Utilizing the Olink Explore 3072 panel, we identified distinct proteomic profiles in iNPH that highlight significant downregulation of synaptic markers and cell-cell adhesion proteins. Alongside vimentin and inflammatory markers upregulation, these results suggest ependymal layer and transependymal flow dysfunction. Moreover, downregulation of multiple proteins associated with congenital hydrocephalus (e.g., L1CAM, PCDH9, ISLR2, ADAMTSL2, and B4GAT1) points to a possible shared molecular foundation between congenital hydrocephalus and iNPH. Through orthogonal partial least squares discriminant analysis (OPLS-DA), a panel comprising 13 proteins has been identified as potential diagnostic biomarkers of iNPH, pending external validation. These findings offer novel insights into the pathophysiology of iNPH, with implications for improved diagnosis.

The brain dense vascular network is essential for distributing oxygen and nutrients to neural cells. The network develops during embryogenesis and leads to the formation of the endothelial blood-brain barrier (BBB). This barrier is surrounded by mural cells (pericytes and vascular smooth muscle cells (VSMCs)) and fibroblasts. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (ECs), VSMCs and fibroblasts follow specific molecular maturation programs over this time period. In particular, ECs acquire P-glycoprotein (P-gP)-mediated efflux capacities. The arterial VSMC network expands, acquires contractile proteins (such as smooth muscle actin (SMA) and myosin heavy chain 11 (Myh11)) and becomes contractile. We also analyzed samples of human brain cortex from the early prenatal stage through to adulthood: the expression of endothelial P-gP increased at birth and Myh11 in VSMCs acts as a developmental switch (as in the mouse) at birth and up to the age of 2 of 5 years. Thus, in both mice and humans, the early postnatal phase is a critical period during which the essential properties of cerebral blood vessels (i.e. the endothelial efflux of xenobiotics and other molecules, and the VSMC contractility required for vessel tone and brain perfusion) are acquired and mature.

BackgroundPatients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 is unable to cross the blood-brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 (Npc2-/-). MethodsSix weeks old Npc2-/- mice were intravenously injected with the AAV-BR1-NPC2 vector. Post-mortem analyses included gene expression analyses, determination of NPC2 transduction in the CNS, and co-detection of cholesterol with NPC2 in neurons. ResultsThe vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector. ConclusionThe data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons.

Exosomes are small extracellular vesicles (sEVs) of [~]30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size ([~]80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute [~]15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of [~]50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.