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Uncovering the role of connexins and Yap in blood brain barrier hyperpermeability and microvascular injury in cerebral amyloid angiopathy

Authors: Muyu Situ 1  and Anuska V. Andjelkovic 2

Affiliations:  1 Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI
2 Department of Pathology and Neurosurgery, University of Michigan, Ann Arbor, MI   

Background (165)
Cerebral  Amyloid  Angiopathy  (CAA)  is  a  cerebral  small  vessels  disease  characterized  by accumulation  of  amyloid-β  (Aβ)  around  the  small  caliber  vessels  (arterioles  and  capillaries).  Structural alterations and loss of integrity in the neurovascular unit/blood-brain barrier (NVU/BBB) in CAA results in “leakier” microvessels, microhemorrhage, increased stroke risk, and cognitive impairment. Although a significant body of evidence has pinpointed several potential mechanisms (i.e.,  neuroinflammation  and  oxidative  metabolism)  to  drive  BBB  injury  in  CAA,  molecular mechanisms remain to be fully elucidated. To determine the profile of microvascular injury in CAA murine model (Tg-SwDI mice), we performed RNA sequencing  analysis on the isolated brain microvessels.  Transcriptome  profiling  revealed  alteration  in  genes  like  gap  junction  proteins, connexin 43 (Cx43) and Cx45, and  Yap1 (Yes-associated protein), a component of the Hippo signaling  pathway  involved  in  remodeling  of  actin  cytoskeleton,  extracellular  matrix,  and regulation of inflammation. Our present study aims to understand how changes and interactions between Cx43/Cx45, and Yap modifies barrier integrity and promotes cerebrovascular injury in the CAA condition.

Methods (57)
Using in vitro (Aβ 1-40  and Aβ 1-42  exposure) and in vivo models  (Tg-SwDI mice), we performed assessment of cell structural changes (immunocytochemistry, confocal microscopy), functional (optical  tweezers  method),  FRAP  assay,  in  vitro  permeability  assay)  and  signaling  events (western blotting and proximity ligation assay) associated with alteration of brain endothelial cells (BECs) mechanics and barrier permeability in CAA settings. 

Results (90)
Consequences of Aβ-afflicted brain microvessels and Aβ exposure in BECs promotes an overall increased protein expression and a redistribution of Yap expression from the cytoplasm to the nucleus, contributing to structural changes, such cellular stiffening and cell permeability. Similarly, Aβ promotes upregulation of Cx43 and Cx45 (i.e., GJs and HCs formation and activity) as well as loss  of  tight  junction  integrity  resulting  in  a  hyperpermeable  barrier.  Inhibition  (via  selective inhibitors) and/or modifications of each component  expression (via siRNA transfection) or whole Yap/Cx43/Cx45 axis alters the cell mechanics and consequently barrier permeability.  Conclusion (36) Collectively, our data suggest that vascular Aβ deposition in CAA pathology promotes injury and junctional  alterations  through  a  Cx43/Cx45/Yap  axis  in  the  brain  endothelium  contributing  to development of CAA associated pathology (i.e., BBB hyperpermeability, and microbleeds).


cARLA: a small molecule cocktail for robust induction of blood-brain barrier properties

Gergo Porkolab 1,2 , Maria Meszaros 1 , Aniko Szecsko 1,2 , Judit P. Vigh 1,2 , Fruzsina R. Walter 1 , Ricardo Figueiredo 3 ,  Ildiko  Kalomista 4 ,  Gaszton  Vizsnyiczai 1 ,  Jeng-Shiung  Jan 5 ,  Fabien Gosselet 6 , Monika Vastag 4 , Szilvia Veszelka 1  & Maria A. Deli 1.
1 Institute  of  Biophysics,  Biological  Research  Centre,  Eötvös  Loránd  Research  Network, Szeged, Hungary
2 Doctoral School of Biology, University of Szeged, Szeged, Hungary
3 GenXPro GmbH, Frankfurt am Main, Germany
4 In vitro Metabolism Laboratory, Gedeon Richter Plc., Budapest, Hungary
5 Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
6 Laboratoire de la Barriére Hémato-Encéphalique (LBHE), Université d’Artois, Lens, France

Correspondence: Gergo Porkolab – <This email address is being protected from spambots. You need JavaScript enabled to view it.>

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of human cerebrovascular diseases and facilitate drug development for the brain.  Yet providing  endothelial  cells  with  the  appropriate  molecular  cues  to  both  retain  a vascular  identity  and  acquire  BBB  characteristics  remains  challenging.  Here  we  present cARLA,  an  easy-to-use  and  affordable  small  molecule  cocktail  that  robustly  induces  BBB properties in vitro. By activating cyclic AMP and Wnt/β-catenin signaling while inhibiting the transforming growth factor beta (TGF-β) pathway, cARLA synergistically enhances barrier tightness in a range of BBB models. We demonstrate that, upon cARLA treatment, human stem cell-derived endothelial cells have lower rates of transcytosis, higher glycocalyx density and increased efflux pump activity with a shift in gene expressional profile towards the in vivo brain endothelial signature. Our work provides mechanistic insight into how endothelial signaling is orchestrated  during  BBB  maturation  and leverages  this  to  advance  the  prediction  of  drug delivery to the human brain.


Tanycytic VEGF receptor signaling in the blood-hypothalamus barrier: a new player in the communication between the brain and the periphery

Ines Martinez-Corral, Emilie Caron, Sreekala Nampoothiri, Vincent Prevot  

Univ. Lille, INSERM, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine brain, Lille Neuroscience and cognition, UMR-S1172, Lille France.  

Correspondence: This email address is being protected from spambots. You need JavaScript enabled to view it.  

Abstract Text
The communication between the brain and the periphery is tightly regulated by the blood brain barrier  (BBB),  in  which  endothelial  cells,  sealed  by  tight  junctions,  and perivascular  cells contribute  forming  the  neurovascular  unit. However,  specific  brain  regions  known as circumventricular organs (CVOs) lack this type of barrier. CVOs are characterized by the presence  of  fenestrated  vascularization  and  the  translocation  of  barrier  properties  to non-endothelial cells.

Among CVOs, the median eminence (ME) is located in the base of the hypothalamus, ventral to the third ventricle and adjacent to  the arcuate nucleus (ARH). The ME is a key interface between  the  neural  and  endocrine  systems  involved  in  the hypothalamic  control  of  energy homeostasis. The presence of a fenestrated endothelium in the ME allows the passive diffusion of blood-borne  molecules  into  the  parenchyma  and  vice-versa. However,  the  passage  of molecules beyond the ME to the ARH or cerebrospinal fluid is tightly regulated by tanycytes. These specialized ependymoglial cells that line the wall of the third ventricle are sealed by tight junctions, forming the blood hypothalamus barrier. Tanycytes send long processes to contact the wall of the fenestrated vessels in the ME, but also of the BBB vessels in the ARH.

In the ME as elsewhere, VEGFR signaling is not specific to blood vessels. Using different approaches, we have observed that a subset of tanycytes expresses VEGFR2, and that in mice, tanycytic VEGFR2 signaling is involved in the maintenance of body homeostasis. A potential role in metabolic diseases such as obesity is also being explored.

Altogether, our results suggest that within tanycytes, vascular-specific pathways are directly involved in the communication between hypothalamus and vasculature, and ultimately between brain and periphery.  


TRIM47 is a crucial regulator of brain endothelial cell functions  

Juliette Vaurs 1 , Claire Peghaire 1 , Valentin Delobel 1 , Romain Boulestreau 1 , Béatrice Jaspard-Vinassa 1 , Carole Proust 1 , Cécile Duplàa 1 , Thierry Couffinhal 1   

1 Biology of cardiovascular disease, University of Bordeaux, Inserm France  

Correspondence: Juliette Vaurs - <This email address is being protected from spambots. You need JavaScript enabled to view it.>  

Cerebral small vessel disease (cSVD) is a leading cause of strokes and a major contributor to vascular  dementia.  Evidence  indicates  that  BBB  dysfunction  may  play  a  significant  role  in vascular  dementia  pathogenesis.  However,  the  molecular  determinants  and  responsible mechanisms  are  still  not  resolved.  Recently,  we  reported,  using  a  human  genome-wide association  study,  an  inverse  correlation  between  the  expression  of  an  ubiquitin  ligase, TRIM47, and extensive cSVD severity. TRIM47 is highly expressed in human and mouse brain endothelial cells (EC), indicative of its putative role at the BBB level.
We demonstrate that TRIM47 knockdown in human brain microvascular EC (HBMEC) impairs BBB properties with an increased permeability, decreased directed migration, and blockage of endothelial sprouting.  TRIM47 loss led to profound alteration in the actin  cytoskeleton organisation assembly via the inhibition of the RhoA/ROCK1/LIMK1 pathway.
By performing RNA-sequencing, we identify that loss of TRIM47 represses NRF2 associated gene expression in HBMEC. NRF2 is an important transcriptional regulator of antioxydant and anti-inflammatory enzymes. It binds to its inhibitor  KEAP1 in the  cytoplasm  under normal conditions. Using proximity labeling (BioID) and co-immunoprecipitation assays, we report an interaction of TRIM47 with KEAP1. Repression of TRIM47 increases KEAP1 level. Finally, we showed that KEAP1 inhibitor treatment restores HBMEC functions impaired by TRIM47 loss. In summary, we report that TRIM47 dysregulation affects EC properties by dysregulating the RhoA/ROCK1 signaling via the NRF2/KEAP1 pathway. It may, per se, reduce stress resistance to EC at the BBB level.


Estrogen signaling contributes to Group B Streptococcal disruption and invasion of brain endothelial cells

Nadine Vollmuth 1  and Brandon J Kim 1   
1 Department of Biological Sciences, University of Alabama, Tuscaloosa AL USA  

Bacterial meningitis is a serious life-threatening infection of the central nervous system (CNS) that occurs when blood-borne bacterial pathogens can disrupt the blood-brain barrier (BBB) and enter the CNS. The BBB is comprised of highly specialized brain endothelial cells (BEC) that serve to protect the CNS from toxins and pathogens while supporting proper brain function. Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and mechanisms of how the BBB fails to protect the CNS during infection remain unclear. We have conducted microRNAseq on BECs either infected with GBS or mock infected and strikingly we found that globally microRNAs are downregulated. Estrogen signaling has been demonstrated to contribute to global microRNA downregulation and we hypothesize that estrogen signaling may play a role in GBS – BEC disruption. Our preliminary findings demonstrate that treatment of BECs with the estrogen receptor (ER) antagonist is sufficient to inhibit GBS invasion of BECs and rescue candidate microRNA expression. Additionally, we find that BECs treated with the ER agonist beta-estradiol is sufficient to reduce microRNA expression, reduce trans-endothelial electrical resistance, and increase rates of bacterial invasion. Our findings suggest that GBS may utilize estrogen signaling to gain access to the CNS. Future work will determine if rescue of microRNAs can restore BBB function during GBS infection. Additionally, we will determine if GBS produces an estrogen-like molecule to induce ER signaling or if GBS induces endogenous ER signaling. We show for the first time, that ER signaling can contribute to GBS invasive disease.


Vascular basement membrane laminins contribute to the functional integrity of the blood vessels

Tushar  Deshpande 1 *,  Jula  Huppert 1 *†,  Sai-Kiran  Samawar 1 ,  Kishan  Kapupara 1 ,  Sharang Ghavampour , Jian Song 1 , Ralf Adams 2 , Hyun-Woo Jeong 2 , Lydia Wachsmuth 3 , Cornelius Faber 3 , Lingzhang Meng 1 , Jens Soltwisch 4  , Melanie-Jane Hannocks 1 , Rupert Hallmann 1  and Lydia Sorokin 1

The  neurovascular  unit  (NVU)  is  the  interconnection  between  the  blood  vessels  and  the surrounding  neurons  and  is  constituted  by  several  cell  layers,  including  endothelium, perivascular cells, astrocytes, and neurons, but also acellular layers that are mainly basement membranes (BMs). BMs underlie endothelium, encase pericytes and smooth muscle cells and mark the pial border and, as such, are in contact with several cellular layers of the NVU. Laminins are essential components of vascular BMs, but little is known about their contribution to the NVU. In the brain, laminin α4 and α5 occur in endothelial and mural BMs. To define their contribution to NVU integrity, we analysed blood vessel enriched, neuron depleted samples from  laminin  α4  knock  out  (Lama4 -/- )  and  endothelial-specific  laminin  α5  knock  out  (Tek-cre:Lama5 -/- ) mice using single cell RNA sequencing. The data was clustered into endothelial, mural and myeloid cell types and unsupervised clustering revealed several sub-types of each category  that  were  altered  in  the  laminin  knock  out  mice.  Lama4 -/-   endothelium  showed enhanced large artery and reduced postcapillary venule sub-clusters. Accordingly, mural cells of Lama4 -/-  mice presented augmented contractile gene signatures. Although devoid of laminin expression, resident myeloid cells exhibited an activated phenotype in Lama4 -/- . Analysis of Tek-cre:Lama5 -/-  samples exhibited the opposite molecular and cellular phenotype to Lama4 -/- , with enhanced postcapillary venule marker expression and reduced contractile nature of perivascular  cells.  However  myeloid  cell  populations  were  not  altered  in  Tek-cre:Lama5 -/-  samples compared to WT controls. Our data suggest direct and indirect roles for vascular laminins  in  the  intercommunication  between  the  endothelium,  mural  cells  and  myeloid populations associated with the arterial wall, all of which are required for the functional integrity of the NVU.

Author affiliations
1 Institute of Physiological Chemistry and Pathobiochemistry and Cells-in-Motion Interfaculty Centre (CiMIC), University of Muenster, Germany; 
2 Department of Tissue Morphogenesis, Max-Planck-Institute  for  Molecular  Biomedicine,  Muenster,  Germany. 
3 Clinical  Radiology, University of Muenster, Germany; 
4 Institute of Hygiene, University of Muenster, Germany


An interplay between HIV infection and cerebrovascular toxicity of methamphetamine

Minseon Park 1 , Andrzej Małecki 2 , and Michal Toborek 1,2
1 Department  of  Biochemistry  and  Molecular  Biology,  University  of  Miami  Miller  School  of Medicine, Miami, FL 33136, USA
2 Institute  of  Physiotherapy  and  Health  Sciences,  The  Jerzy  Kukuczka  Academy  of  Physical Education, Katowice 40-065, Poland  

Corresponding Author: Michal Toborek; This email address is being protected from spambots. You need JavaScript enabled to view it.  

A substance use disorder (SUD) is a well-recognized risk factor for HIV-1 infection; however, little is known about molecular interactions between psychoactive substances and HIV-1 in the brain, and how they impact their long-term effects. The problem is important because SUD, including methamphetamine (METH) use, raises the risk of contracting or transmitting HIV-1 not only for individuals who inject the drug, but also for noninjecting users, as it is associated with an increase in risky sexual behavior. Moreover, METH use, either alone or in combination with other drugs, is associated  with  failure  of  viral  suppression.  People  living  with  HIV-1  (PLWH)  who  engage  in METH  use  display  exacerbations  in  key  pathophysiologic  processes  that  are  linked  to  faster clinical HIV-1 progression. Indeed, the combination of HIV-1 infection and METH dependence causes  more  profound  neurocognitive  deficits  and  structural  brain  abnormalities  than  either condition alone. METH use increases genomic activities to become transcriptional events related to neurodegeneration, dopaminergic deficits and decreased cognitive and executive functions. Our research has focused on the central hypothesis that the immunomodulatory impact of METH on BBB pericytes drives their HIV-1 infection and alters their interactions with neural progenitor cells  (NPCs),  which  are  located  in  perivascular  niches  in  a  close  proximity  to  the  BBB.  We identified that both BBB pericytes and NPCs can be infected with HIV and  characterized  the signaling pathways by which exposure to METH and HIV contributes to aberrant neurogenesis, migration, and proliferation of NPCs. Overall, this research contributes to a better understanding of the influence of psychoactive substances on pericyte HIV-1 reservoirs in the CNS in order to design future therapies for reservoir clearance and a cure for HIV-1, especially for individuals living with HIV-1 who experience a SUD.
Funding: Supported by the National Institutes of Health (NIH) grants DA044579 and DA050528, and by the National Science Centre (NSC) grant 2019/35/B/NZ7/03155.


Extracellular vesicle-mediated transfer of functional molecules from the periphery to the brain

Dr. Stefan Momma, Neurological Institute (Edinger Institute) Goethe University Hospital Frankfurt am Main, Germany. This email address is being protected from spambots. You need JavaScript enabled to view it.  

Extracellular vesicles (EVs) are emerging as important carriers for intercellular and interspecies communication and have been implicated in many biological processes. However, due to their small size and the difficulties in manipulating them, data on EV biology have thus far been based mainly on in vitro or indirect in vivo evidence. Therefore, the extent and pathogenic role of EV signaling in vivo, particularly with regard to the transfer of functional molecules, remains poorly understood. To overcome some of the major shortcomings in this field, we introduced the Cre-Lox system to establish a method to trace the functional transfer of RNA or protein by EVs in vivo. We showed that the expression of Cre recombinase in blood cells and bacteria leads to the release of EVs containing Cre mRNA and proteins, respectively. As a result, in mice with a Cre reporter background, the uptake of Cre-containing EVs leads to an irreversible induction of marker gene expression. This allowed us to identify a novel route of communication between the immune system, gut bacteria, and the brain, crossing the intestinal epithelial and blood-brain barriers.


Placenta-derived extracellular vesicles: their unique characteristics of the blood-brain barrier transport  

Masanori Tachikawa 1 , Mai Inagaki 1 , Hinori Sano 2 , Momoko Sato 2 , Hidetaka Kosako 3 , Kenichi Funamoto 4  

1 Graduate  School  of  Biomedical  Sciences; 
2 Graduate  School  of  Pharmaceutical  Sciences;
3 Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan; 
4 Institute of Fluid Science, Tohoku University, Sendai, Japan  

Correspondence: Masanori Tachikawa – <This email address is being protected from spambots. You need JavaScript enabled to view it.>  

The placenta,  an organ specific for pregnant women, secrets  the unique nano-size particles which we call placenta-derived extracellular vesicles (pEVs). The pEVs encapsulate nucleic acid,  e.g.,  miRNA,  and  functional  proteins  as  message  substances  and  play  a  role  in  the placenta-to-maternal  organs  signal  transductions.  An  interesting  report  has  shown  that pregnancy causes a reduction in the brain gray matter region subserving social cognition (Nat Neurosci  20:287-296,  2017).  This  implies  that  pEVs  could  mediate  the  placenta-to-brain delivery of the message substances such as miRNA beyond the blood-brain barrier (BBB). In support of this notion, we found that the placenta-related miRNAs are present in the pregnant mouse brain as well  as in blood-circulating extracellular vesicles. Thus,  the purpose of  the present study was to clarify the pEVs transport at the BBB. The pEVs were obtained from the human placental trophoblast cells (BeWo cells) by ultracentrifugation. The three-dimensional human brain microvasculature model was our originally constructed on a microfluidic device. We succeeded in visualizing the pEVs which were transported to the brain parenchymal cells across the brain microvasculature on a microfluidic device. We also identified the virus receptor as the potent transport system of pEVs at the BBB by proteomics- and gene knockout-based studies. In this symposium, I will introduce our recent data on the uniqueness of pEVs and the transport characteristics of pEVs at the BBB, which have been clarified by human BBB on-a-chip and proteomics. I will also propose a concept of pEVs-mediated placenta-to-brain signal transduction via the BBB.


Multi-omics characterization of the blood-brain barrier in molecular groups of ependymoma 

Julia K. Sundheimer 1,2 , Julia Benzel 1 , Aniello Federico 1 , Maximilian Knoll 6 , Cornelia Gansert 1,2 , Britta Statz 1,2 , Christian Seitz 4,5 , Marcel Kool 1 , Stefan M. Pfister 1,3 , Kendra K. Maaß 1,3 , Kristian W. Pajtler 1,3 
[1]     Hopp Children’s Cancer Center Heidelberg (KiTZ) and Division of Pediatric Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
[2]     Faculty of Biosciences, Im Neuenheimer Feld 234, 69120 Heidelberg University, Germany
[3]     Department of Pediatric Hematology, Oncology and Immunology, Heidelberg University Hospital, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany 
[4]     DFG Cluster of Excellence 2180 'Image-guided and Functional Instructed Tumor Therapy' (iFIT), University of Tuebingen, Tuebingen, Germany.
[5]     Department of Pediatric Hematology and Oncology, University Hospital Tuebingen, Tuebingen, Germany.
[6]     Division of Molecular & Translational Radiation Oncology, Heidelberg Ion-Beam Therapy Center (HIT), Heidelberg University Hospital (UKHD), Heidelberg, Germany; German Cancer Consortium (DKTK) Core-Center, German Cancer Research Center (DKFZ), Heidelberg, Germany; Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Oncology (NCRO), German Cancer Research Center (DKFZ), Heidelberg University Hospital (UKHD), Faculty of Medicine (MFHD) of the Heidelberg University, and Heidelberg Ion-Beam Therapy Center (HIT), Heidelberg, Germany; CCU Translational Radiation Oncology, CCU Radiation Oncology, CCU Neurooncology, National Center for Tumor Diseases (NCT) German Cancer Research Center (DKFZ), Heidelberg University Hospital (UKHD), Heidelberg, Germany; Departments of Neurology, Neurosurgery and Radiation Oncology, Heidelberg University Hospital (UKHD), Heidelberg, Germany.  

Correspondence: This email address is being protected from spambots. You need JavaScript enabled to view it.  

Ependymoma (EPN) represents the third most common pediatric CNS tumor. While recurrence rates remain high, systemic therapies have so far failed to lead to clinical benefit. A better understanding of pathophysiological blood-brain barrier (BBB) characteristics represents an important component in developing effective (pre-)clinical trials. 

Our study seeks to increase knowledge of molecular EPN group-specific BBB compositions as a proof-of-concept for other brain tumor entities. Furthermore, we explore the correlation between BBB characteristics and their functional impact to adapt an established in silico model that currently predicts drug penetration over the healthy BBB.

T-distributed stochastic neighbor embedding (tSNE)-based clustering analyses using the most relevant  tight  junction  and  transporter  gene  sets  revealed  distinct  molecular  EPN  group-specific expression patterns. While PDX models (n=20) showed high similarity with patient tumor samples, IUE mouse models (n=2) did not fully recapitulate these BBB characteristics. Single-cell analyses and spatial mapping of protein abundance allowed dissection of BBB gene expression patterns in endothelial cells (e.g. Claudin5). Functional validation on protein level showed that coherence of RNA and protein is BBB gene-dependent.

The  differences  in  BBB  markers  between  molecular  EPN  groups  may  partly  explain  drug resistance of aggressive EPN as especially ZFTA fusion-positive tumors are characterized by high tight junction expression suggestive of low BBB permeability. Our multi-omics approach is intended to develop a score that further complements our established in silico prediction tool for BBB drug penetration. These findings will be validated in preclinical studies while molecular BBB characterization will be further expanded to other brain tumor entities.


Unraveling the role of biological barriers in the development of chemotherapy-induced peripheral neuropathy   

Yang Hu 1,2 , Milda Girdenyté 1 , Frida Bällgren 1 , Lieke Roest 1 , Iida Liukkonen 1 , Maria Siskou 1 , Margareta Hammarlund-Udenaes 1 , and Irena Loryan 1 *  

1  Translational Pharmacokinetics/Pharmacodynamics Group, Department of Pharmacy, Faculty of Pharmacy, Uppsala University, Box 580, SE-751 23, Uppsala, Sweden
2  Current affiliation: Discovery ADME-Drug discovery sciences, Boehringer Ingelheim RCV GmbH & Co KG, A-1121, Vienna, Austria  

*  Correspondence to Irena Loryan: This email address is being protected from spambots. You need JavaScript enabled to view it.    

Although drug distribution into the central nervous system (CNS) has been extensively studied, little is known on how drugs are transported into the peripheral nervous system (PNS). It has been shown that blood-tissue barriers in the CNS and PNS differ in their morphological structures, including tight junctions and transporter expression profiles. However, the functional role of PNS barriers on target-site exposure compared to CNS barriers remains poorly understood, requiring systematic neuropharmacokinetic (neuroPK) evaluations. This study aimed to quantitatively assess and compare the extent of drug transport across the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), blood-nerve barrier (BNB), blood-dorsal root ganglia barrier (BDB), and parenchymal cellular barriers in respective tissues in rats using a set of 11 small molecular weight drugs. Two key neuroPK parameters, namely unbound tissue-to-plasma concentration ratio (K p,uu ) and unbound intracellular-to-extracellular concentration ratio (K p,uu,cell ), were estimated using the Combinatory Mapping Approach. We found that the extent of BDB and BNB transport was significantly greater than that of BBB and BSCB transport, with the difference in K p,uu  up to 635-fold observed for vincristine. The extent of drug transport at the BDB was overall the highest. The discrepancy in cellular transport properties was also observed between the investigated PNS and CNS tissues without any universal pattern. This study sheds light on the key features of unbound drug disposition into the dorsal root ganglia and the sciatic nerve vs. brain and spinal cord, providing invaluable insight into the target-site pharmacological/toxicological effects and development of chemotherapy-induced peripheral neuropathy.  
This project is funded d by EU IMI2 project NeuroDeRisk, grant# 821528.


A sticky situation: the influence of microvessel mechanics on cerebral malaria pathogenesis 2. Brain barriers in disease 

Matt Govendir 1, 2 , Rory Long 1 , Alba Diz Muñoz 2  , Maria Bernabeu 1 
1  EMBL Barcelona, Barcelona, Spain; 
2  EMBL Heidelberg, Heidelberg, Germany  

Correspondence: Matt Govendir - <This email address is being protected from spambots. You need JavaScript enabled to view it.>

Cerebral malaria is the  most severe  and fatal consequence of malaria infection and occurs when Plasmodium falciparum-infected red blood cells (iRBCs) become sequestered in the brain vasculature, leading to vascular obstruction and disruption of the blood brain barrier (BBB). Interactions between iRBCs  and  cerebral  endothelial  cells  that  line  the  blood  vessels  occur  under  dynamic  mechanical conditions  due  to  changing  flow  conditions  within  vessels  and  heterogeneity  in  tissue  stiffness surrounding the vessels.  We utilise both  2D brain endothelial monolayers and  3D bio-engineered cerebral blood vessels that allow for the visualisation of iRBC binding, disruption of flow and BBB breakdown in real-time. By customising both the matrix composition, geometry and flow rate in these models we can precisely tune the mechanics of our system to observe their role in endothelial cell function and cerebral malaria pathogenesis. We reveal that when brain endothelial cells and vessels are  cultured  with  iRBC  products  they  demonstrate  increased  permeability  and  altered  adherens junctions and increased actomyosin contractility, ICAM-1 expression. Furthermore, when cultured on collagen I coated matrices or substrates, brain endothelial cells show reduced disruption by these iRBC toxins.  Our  results  demonstrate  that  both  intracellular  mechanics  (cytoskeletal  organisation)  and extracellular mechanics (cell-matrix interactions) play critical roles in regulating brain endothelial cell and BBB function in both health and disease. Further work is currently ongoing to directly measure changes  to  endothelial  monolayers  and  3D  vessel  mechanics,  via  atomic  force  and  Brillouin microscopy, when cultured on substrates of varying stiffness and after iRBC addition.


Open pathways for cerebrospinal fluid outflow at the cribriform plate

Irene  Spera1,  Nikola  Cousin2,  Miriam  Ries2,  Anna  Kedracka2,  Alina  Castillo1,  Simone Aleandri3,  Mykhailo  Vladymyrov1,  Josephine  A.  Mapunda1,  Britta  Engelhardt1,  Paola Luciani3, Michael Detmar2 and Steven T. Proulx1 
1 Theodor Kocher Institute, University of Bern, Bern, Switzerland,
2 Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland, and
3 Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland 

Correspondence: Steven T. Proulx. Email:  This email address is being protected from spambots. You need JavaScript enabled to view it.   

Routes  along  the  olfactory  nerves  crossing  the  cribriform  plate  to  lymphatic  vessels  in  the  nasal submucosa are critical cerebrospinal fluid (CSF) outflow pathways. However, it remains unclear how fluid pathways along the nerves connect to the lymphatic vessels and where the arachnoid barrier is breached. Here, we anatomically defined the connections between the subarachnoid space (SAS) of the central nervous system and the lymphatic system.  

PEGylated fluorescent microbeads were infused into the CSF space of Prox1-GFP reporter mice to identify the CSF outflow pathways. A labeled anti-CD31 vascular antibody was infused into the cisterna magna to study the connections to the lymphatic vessels in the olfactory region. In some samples, we did immunofluorescence staining on decalcified sections to detect the arachnoid barrier.

PEG microbeads were within lymphatic vessels in the nasal submucosa and the lumen of lymphatic vessels that crossed the cribriform plate alongside the olfactory nerves. The labeled antibody revealed a continuous functional network of lymphatic vessels draining CSF from the SAS through the nasal submucosa. In addition, we observed discontinuity of the arachnoid barrier at the olfactory region.  

Micron-sized PEG beads are suitable for studying CSF bulk flow and clearance pathways. We observed direct and open connections from the CSF space to the lymphatic vessels crossing the cribriform plate and in the nasal submucosa. Finally, the discontinuous distribution of the arachnoid barrier in this area could explain how CSF within the SAS accesses the lymphatic system.