Final Report Summary - JUSTBRAIN (Blood-brain barrier junctions as targets for paracellular drug delivery to the brain)
Executive Summary:
To maintain homeostasis of the brain, passage of potentially harmful substances from the blood-stream into the brain is restricted by the highly specialized vascular barrier, the blood-brain barrier (BBB). In this neuroprotective role, the BBB also hinders the delivery of many potentially important diagnostic and therapeutic drugs to the central nervous system (CNS). Current estimates are that only 2% of small molecule drugs and negligible amounts of large molecule drugs in clinical use cross the BBB. This has dramatically slowed the development of pharmacotherapies and immunotherapies in brain diseases. Because most drugs cannot penetrate the BBB, the treatment of certain diseases such as primary brain tumors or brain metastases is presently reduced to symptomatic and palliative measures. Previous studies have exploited specific transport or receptor-mediated transcytosis systems, selectively expressed by brain endothelial cells to ensure the effective delivery of nutrients from the blood to the brain and the rapid removal of toxic metabolites from the brain for drug delivery into the brain with limited success. Making use of the recently increased knowledge in the molecular composition of endothelial cell tight junctions (TJs), sealing the spaces between the BBB endothelial cells, JUSTBRAIN was dedicated to specifically explore and develop strategies targeting individual junctional proteins or their regulation for a controlled and transient – thus safe - opening of BBB junctions allowing for the delivery of large molecular drugs into the brain through these open junctions.
To achieve this goal JUSTBRAIN has integrated basic research teams from Italy, France, Germany and Switzerland that have significantly contributed to the recent discoveries on the molecular architecture of endothelial junctions and to the emerging knowledge on the expression and regulation of these junctional complexes in endothelial cells in general and in brain endothelium specifically. The consortium is complemented with a pharmaceutical enterprise (F. Hoffmann-La Roche Ltd., Basel, CH) as well as a technology partner for project management (tp21, D) .
JUSTBRAIN has focused on the role of cell-to-cell junctions in the control of BBB permeability. The workflow was organized in eight work packages (WPs), which were dedicated to identify therapeutic targets for the manipulation of BBB cell-to-cell junctions and to develop and test feasibility of novel strategies for delivery of large drugs through BBB cell-to-cell contacts. While the first two WPs were dedicated to characterize the molecular composition of the different junctional complexes between brain endothelial cells with the idea to define suitable targets for manipulation of BBB junctions, the 3rd WP investigated transcription factors regulating the BBB cell-cell junctions. Three additional WPs focused on testing and verifying the strategies identified in the other WPs for paracellular delivery of large drugs across the BBB in vitro and in vivo in health and disease. Finally, two WPs were dedicated to the successful management and networking and dissemination of results within and beyond JUSTBRAIN.
Besides the establishment for numerous novel approaches that can be explored in the future for opening BBB TJs for the delivery of large drugs into the CNS, the JUSTBRAIN collaborative research has achieved its major goal by identifying with the canonical Wnt/-catenin signaling pathway one molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large drugs into the brain in vivo during health and neuroinflammation.
JUSTBRAIN has therefore achieved breakthroughs in the development of paracellular bypasses of the BBB for the safe delivery of large drugs into the brain.
Project Context and Objectives:
There is a lack of therapies for most neurological disorders ranging from rare, however, fatal diseases such as lysosomal storage diseases and primary brain tumors to major public health concerns such as brain metastases, multiple sclerosis, stroke or Alzheimer’s disease. In addition to the development of effective drugs, the delivery of diagnostic or therapeutic compounds into the brain has become a major challenge in successfully treating these diseases. To maintain homeostasis of the brain, passage of potentially harmful substances from the blood stream into the brain is restricted by the highly specialized vascular barrier, the blood-brain barrier (BBB). In this neuroprotective role, the BBB also hinders the delivery of many potentially important diagnostic and therapeutic drugs to the central nervous system (CNS). Current estimates are that only 2% of small molecule drugs and negligible amounts of large molecule drugs in clinical use cross the BBB. This has dramatically slowed the development of pharmacotherapies and immunotherapies in brain diseases. Because most drugs cannot penetrate the BBB, the treatment of diseases as primary brain tumors or brain metastases is presently reduced to symptomatic and palliative measures.
JUSTBRAIN research focus
The BBB inhibits the free trans-cellular passage of hydrophilic molecules by an extremely low pinocytotic activity and their paracellular diffusion by an elaborate network of complex TJs between its endothelial cells. Specific transport or receptor-mediated transcytosis systems, selectively expressed by brain endothelial cells to ensure the effective delivery of nutrients from the blood to the brain and the rapid removal of toxic metabolites from the brain have been exploited for drug delivery. Unspecific opening of the endothelial junctions as performed in selected clinics today, i.e. by hyperosmotic shock, bears a high risk of toxicity as it is accompanied by the influx of substances other than the drug of interest into the brain and may thus culminate in global adverse drug reactions. Therefore, controlled and transient opening of BBB TJs is required for safe paracellular drug delivery into the brain.
JUSTBRAIN was dedicated to specifically explore and develop strategies targeting individual junctional proteins for a controlled and transient – and thus safe - opening of BBB junctions allowing for the delivery of large drugs into the brain. This approach was considered particularly difficult so far due to the poor knowledge of BBB junction organization and, most importantly, for the lack of treatments able to target these structures specifically and reversibly. Prior to the beginning of their collaboration, the groups of the JUSTBRAIN consortium had collected a large set of data during their previous work, which offered a unique platform for the development of novel and targeted therapeutic approaches to manipulate paracellular BBB permeability. In order to proceed in a fast and competitive way, these groups combined their complementary approaches, which included in vitro and in vivo tests and animal models of CNS pathologies, (e.g animal models for multiple sclerosis) where BBB opening may be therapeutic. The wide range of expertise offered by this consortium was unique in Europe for the particular approach of targeting BBB junctions for paracellular drug delivery into the brain.
In pursuit of this goal JUSTBRAIN aimed to identify an entirely novel platform of drugable molecular targets to bypass the BBB and thereby improve delivery of large molecules into the brain and thus expand on diagnostic and therapeutic possibilities for neurological disorders.
To achieve this goal JUSTBRAIN
• Defined the best known molecular targets of BBB junctions (trans-membrane junctional adhesion proteins and/or their intracellular binding partners) by applying available inhibitors and therapies in health and disease
• Characterized novel junctional adhesion proteins and/or their intracellular binding partners in health and disease
• Characterized the transcriptional control of BBB junction maintenance in health and disease.
• Transiently increased or decreased expression of other/novel junctional proteins (JAMs, L1) at the BBB in vitro and in vivo
• Modulated the extracellular homophilic or heterophilic interaction of tight junction proteins, i.e. claudins at the BBB in vitro and in vivo
• Choose feasible approaches for paracellular drug delivery to be further developed for potential clinical development
Project Results:
JUSTBRAIN has made an in depth analysis of the molecular composition of the cell-to-cell contact complexes in between brain microvascular endothelial cells establishing adherens junctions (AJs) and tight junctions (TJs) at the blood-brain barrier (BBB) in vitro and in vivo. In addition it characterized the presence of molecules localized in cell-to-cell contacts of the BBB outside of the organized junctions.
Analysis was performed in mouse models investigating healthy mice and mouse models for Alzheimer’s disease, multiple sclerosis, brain tumors and vascular malformations modelling cerebral cavernous malformations. Additional analysis was performed in a number of different mouse and human in vitro models for the BBB. In this context JUSTBRAIN specifically
• Described the molecular organization of AJs and TJs in endothelial cells in primary cultures from mouse brain and in mouse and human endothelial cell lines of brain origin
• Established culture conditions to induce stabilization of AJs and TJs in brain endothelial cells in culture
• Confirmed the composition of endothelial junctions in brain samples and in retinae from mice, transgenic and pathological models
• Identified one cytoplasmic component of AJs as crucial for the maintenance of barrier properties and for the organization of tight junctions and established a BBB transcript signature that defines BBB TJs.
Furthermore JUSTBRAIN established novel transgenic mouse models for mimicking human diseases affecting the BBB and allowing for deletion or increased expression of specific molecules localized in BBB AJs (CCM1, -2 and -3) and TJs and cell-to-cell contacts.
Using these models JUSTBRAIN identified transmembrane components of AJs and TJs that are crucial for enhancing the barrier properties of brain endothelial cells.
Cerebral cavernous malformations (CCM) are vascular malformations of the (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. CCM lesions are formed by enlarged and irregular blood vessels. In addition, as the CCM complex can associate with adherens junctions an important role in junctional integrity of the endothelium is suggested. Indeed loss of function of any CCM protein strongly affects AJ organization and AJ permeability. Most importantly, vascular fragility is markedly increased, resulting in lesions that frequently bleed and cause headache, seizures and eventually hemorrhagic stroke in the affected patients.
JUSTBRAIN therefore established by deleting CCM1, or -2 or -3 mouse models for CCM as a tool to identify signaling pathways governing adherens and tight junctions in endothelial cells of the brain. JUSTBRAIN showed that postnatal deletion of any of the three CCM genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. We showed that endothelial-specific disruption of the CCM1 gene in mice induces Endothelial-to-mesenchymal transition (EndMT), which contributes to the development of vascular malformations. CCM-1-ablated endothelial cells showed upregulation of endogenous BMP6 which activated the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway leading to EndMT. We could demonstrate that inhibition of TGF-β and BMP singnaling pathways prevented EndMT both in vitro and in vivo and reduced the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. In further studies, we have observed that the first event in the induction of the EndMT phenotype following loss of CCM3 gene is the activation of Wnt/-catenin signalling. This is then followed by activation of the TGF-/BMP. Pharmacological inhibition of -catenin transcriptional signalling inhibited the progression of vascular malformations in vivo. Therefore, a cross-talk exist between two crucial signalling pathways, that both regulate terminal differentiation in several cellular context.
CCM is one of the first conditions described that shows that altered junctional composition of BBB cell-to-cell junctional organization can affect vascular morphogenesis and can be the cause of severe pathologies. Thus, exploration of the CCM complex and signaling pathways in JUSTBRAIN established novel insight on a molecular complex critically involved in controlling junctional integrity and hence on the future possibility for targeting of BBB junctional complexes for therapeutic opening of BBB TJs for drug delivery.
On the other hand JUSTBRAIN accumulated a vast amount of “negative” data demonstrating that although expressed in BBB cell-to-cell junctions numerous molecules can be deleted without loss of function for BBB junctional integrity. Examples include the family of junctional adhesion molecules (JAMs), which within JUSTBRAIN were identified to not provide suitable target molecules for transient opening of BBB TJs. JUSTBRAIN has established that, unlike in other vascular barriers, the JAM family is not critically involved in regulating the integrity of BBB TJs and AJs in vivo and in vitro. Specifically, mouse models with deletions for JAM-A, JAM-B or JAM-C and endothelial overexpression of JAM-C did not result in loss of barrier functions at the BBB in vitro and in vivo. At the same time JAMs do influence leukocyte trafficking across the BBB. Therefore JAM-A, JAM-B and JAM-C are unsuitable targets for regulating BBB TJs and AJs. Although this is a negative finding it emphasizes that BBB TJs are different from TJ in other vascular barriers, which might allow for BBB-specific manipulations of TJ integrity.
Further molecules that could be excluded as targets for manipulating BBB TJ integrity to allow for the delivery of large molecular drugs across the BBB were: ICAM-1, CD73 and L1.
Using mouse models with increased or deleted expression of claudins (Clds), thus altering BBB TJ composition, JUSTBRAIN established first of all that Cld-1 is not expressed at the BBB. However, sealing of BBB TJs by ectopic expression of Cld-1 in BBB endothelium reduces BBB leakiness and ameliorates clinical disease in an animal model for multiple sclerosis, thus showing that modulation of Cld localization in BBB TJs is critical for controlling BBB TJ permeability. Using our in vitro model of human BBB, the hCMEC/D3 cell line, we identified a new partner of Cld5 that regulates TJ integrity: the Guanine nucleotide binding protein Gi2 (Luissint et al. JCBFM 2012).
At the beginning of JUSTBRAIN one of the most promising strategies for transient opening of the BBB TJs was therefore considered to be the modulation of TJ integrity by modulatory targeting of the homophilic or heterophilic interactions of claudins in cis and trans by the use of Cld-targeting peptides. JUSTBRAIN demonstrated for the first time that the first extracellular loops (ECL1) of Cld1 and Cld5 are involved in homo- and heterophilic interactions between classic Clds. The interactions were shown to be redox-sensitive, which is of relevance for pathological conditions related to oxidative stress.
Inhibition of these homo- and heterophilic Cld-interactions therefore seemed a promising approach for the transient opening of BBB TJs for the delivery of large drugs across the BBB. To this end, we developed Cld-targeting peptides based on the amino acid sequences of the ECLs of a number of Clds and characterized and tested their ability to transiently open BBB TJs. Cld-targeting peptides related to the first extracellular loop of Cld1 (especially peptides of the C-terminal half of Cld1-ECL1, e.g. mC1C2) were found to increase paracellular permeability of in vitro mouse models of the BBB in a concentration- and time-dependent manner without exerting toxic effects on BBB mouse endothelial cells in culture. However, it has to be mentioned that these peptides were found to be toxic to the human brain endothelial cell line hCMEC/D3. The most significant results were obtained with mC1C2. Modifications at the C-terminus may even improve the BBB TJ opening properties of mC1C2. mC1C2 was found to bind to Cld1 and to Cld5 with nanomolar binding constants explaining how it can target BBB endothelium lacking Cld1 expression but showing high expression of Cld5. The mechanism of action defined was that mC1C2 internalized Cld1 via the clathrin pathway which, in turn, caused delocalization of classic Clds from the membrane to cytosol by a mechanism not fully understood. In general, mC1C2 therefore seems to reversibly modulate tissue barriers via specific and unspecific binding to the ECL1 of classic Clds, inducing down-regulation of membranous Clds.
Unfortunately, this approach completely failed in vivo where JUSTBRAIN could demonstrate that systemic injection of Cld-targeting peptides that allowed for transient opening of BBB TJs in vitro exerts toxic effects leading to sudden death and induction of seizures in injected animals. Respecting the 3R rules of animal experimentation JUSTBRAIN decided that more in depth in vitro research is required to fully understand the mode of action of these peptides before aiming for a detailed analysis of their potential for transient opening of BBB TJs in vivo.
Thus JUSTBRAIN could exclude numerous approaches, e.g. targeting the junctional adhesion molecules or claudins with Cld-targeting peptides either due to the lack of a specific function of these molecules on modulating paracellular permeability at the BBB or due to severe toxic side effects exerted by these approaches.
However, an alternative approach might be the targeting of Clds expressed at the BBB by monoclonal antibodies specifically recognizing the extracellular domains of Clds. Such antibodies have been developed in JUSTBRAIN and might provide a safer alternative for interrupting the homophilic and heterophilic Cld interactions in cis and trans for the transient opening of BBB TJs.
When investigating the role of Wnt/β-catenin signalling in maintaining integrity of BBB TJs we discovered that the non-canonical Wnt/PCP pathway is also involved in the regulation of BBB TJ integrity and manipulation of this signalling pathway translates in opening of BBB TJs. Specifically, we showed that inhibition of the non-canonical Wnt/PCP pathway by knocking-down PAR3, a component of the PAR3/PAR6/PKC complex, increased the permeability of in vitro models of the BBB. Interestingly, this alteration in permeability was limited to small compounds (LY and 4kDa-dextran) while no change of the BBB permeability for high MW compounds (70kDa- Dextran and FITC-GAM Antibody) could be observed. Thus, targeting the non-canonical Wnt/PCP pathway, discovered in JUSTBRAIN to modulate BBB TJs, did allow the reproducible opening of the BBB for the passage of small but not large molecules across the BBB. These findings are potentially of clinical importance, suggesting that controlling this pathway might be considered at least as an adjunct strategy for the regulation of BBB permeability.
Excluding on the one hand numerous approaches for the therapeutic manipulation of BBB TJs in vivo, JUSTBRAIN reached its overall goal, which was to define at least one molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large drugs into the brain in vivo during health and neuroinflammation.
The canonical Wnt/-catenin signalling pathway was shown to be relevant for BBB formation and BBB maintenance in the mouse in vivo and in vitro.
Specifically JUSTBRAIN demonstrated that
• the Wnt/-catenin signalling pathway is also involved in the maturation of a human in vitro BBB model – hCMEC/D3 - by inducing the expression of claudin-3 and probably claudin-5
• catenin transcription is required for BBB maintenance in brain ECs in vivo in transgenic mouse models of catenin gain-of-function and loss-of-function mutations thus demonstrating that manipulating Wnt/β-catenin signalling in vivo in transgenic mouse models allowed to regulate the permeability of BBB TJs
• Wnt1, but not control- or Dkk1- expressing GL261 glioma cells showed maintained barrier characteristics of tumour vessels in vivo.
• activation of catenin transcription by Wnt3a as well as by LiCl or BIO increased barrier properties and expression of TJ genes (Cld-3 and -5) in mouse and human in vitro models of the BBB.
• By identifying novel binding partners of AJ (e.g. CCM1, CCM3) and regulators of the Wnt/-catenin signalling pathway controlling BBB junctional integrity that this pathway can be even further explored for safe pharmacological targeting with the goal to open BBB TJs for drug delivery.
• that barrier properties of in vitro BBB models can be improved by stimulating the Wnt/β-catenin signalling, allowing for the establishment of novel and easy to handle in vitro BBB models for high throughput screening
• that small molecular compounds can be used to pharmacologically inhibit the transcriptional activity of catenin in brain ECs in order to transiently open the BBB tight junctions in vitro and in vivo
Thus JUSTBRAIN has identified the canonical Wnt/-catenin signalling pathway as the most promising molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large molecular drugs into the brain in vivo during health and neuroinflammation.
Potential Impact:
By discovering the canonical Wnt/-catenin signalling pathway as a molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs and potentially of the metabolic BBB, allowing for the delivery of large molecular drugs into the brain in vivo during health and neuroinflammation, JUSTBRAIN has established a breakthrough in the development of paracellular bypasses of the BBB for the safe delivery of large molecular drugs into the brain.
The major impacts of JUSTRBRAIN are:
Scientific and clinical impacts
• The demonstration of proof-of-principle of a novel therapeutic strategy for drug delivery across the BBB, which may be developed into future clinical applications.
• A common set of research tools and technologies, which may lead to therapeutic applications in many different pathological conditions.
• The possibility to increase availability of effective medical products to CNS. Many effective drugs showed limited success in the clinic due to their limited capacity to effectively cross BBB.
• Generate a more profound understanding of the biology of the BBB permitting a better design of therapeutic strategies through the cell biology to pre-clinical feedback loop applied. Most products are still in early stage of development due to the absence of easy, low cost, clear-cut assays for BBB penetrance.
• Identify approaches to generate high value and high impacting therapeutic agents, which European companies can effectively exploit.
European Strategic Impacts
• The development of a high-value new technology for the delivery of large drugs into the brain
• To boost the European biotechnology industry, and contribute to developing standard protocols to test drugs for crossing the BBB
• Improve competitiveness of European pharmaceutical companies with highly innovative products that will permit European companies to become global players.
Socio economic impacts
• The development of an European capability in diagnosis and therapy of the increasing number of neurological disorders in an ageing population, which are currently either untreatable or with insufficient therapy (brain tumors, multiple sclerosis, stroke, Alzheimer's Dementia etc.) with the potential for combating rising health costs.
• Facilitate the creation of novel therapeutics, which will decrease the burden for European citizens and health agencies of neurodegenerative diseases.
List of Websites:
Project website: www.JUSTBRAIN-fp7.eu
Coordinator contact: Prof. Britta Engelhardt, Theodor Kocher Institut Bern, University of Bern, Switzerland, E-Mail: bengel@tki.unibe.ch
To maintain homeostasis of the brain, passage of potentially harmful substances from the blood-stream into the brain is restricted by the highly specialized vascular barrier, the blood-brain barrier (BBB). In this neuroprotective role, the BBB also hinders the delivery of many potentially important diagnostic and therapeutic drugs to the central nervous system (CNS). Current estimates are that only 2% of small molecule drugs and negligible amounts of large molecule drugs in clinical use cross the BBB. This has dramatically slowed the development of pharmacotherapies and immunotherapies in brain diseases. Because most drugs cannot penetrate the BBB, the treatment of certain diseases such as primary brain tumors or brain metastases is presently reduced to symptomatic and palliative measures. Previous studies have exploited specific transport or receptor-mediated transcytosis systems, selectively expressed by brain endothelial cells to ensure the effective delivery of nutrients from the blood to the brain and the rapid removal of toxic metabolites from the brain for drug delivery into the brain with limited success. Making use of the recently increased knowledge in the molecular composition of endothelial cell tight junctions (TJs), sealing the spaces between the BBB endothelial cells, JUSTBRAIN was dedicated to specifically explore and develop strategies targeting individual junctional proteins or their regulation for a controlled and transient – thus safe - opening of BBB junctions allowing for the delivery of large molecular drugs into the brain through these open junctions.
To achieve this goal JUSTBRAIN has integrated basic research teams from Italy, France, Germany and Switzerland that have significantly contributed to the recent discoveries on the molecular architecture of endothelial junctions and to the emerging knowledge on the expression and regulation of these junctional complexes in endothelial cells in general and in brain endothelium specifically. The consortium is complemented with a pharmaceutical enterprise (F. Hoffmann-La Roche Ltd., Basel, CH) as well as a technology partner for project management (tp21, D) .
JUSTBRAIN has focused on the role of cell-to-cell junctions in the control of BBB permeability. The workflow was organized in eight work packages (WPs), which were dedicated to identify therapeutic targets for the manipulation of BBB cell-to-cell junctions and to develop and test feasibility of novel strategies for delivery of large drugs through BBB cell-to-cell contacts. While the first two WPs were dedicated to characterize the molecular composition of the different junctional complexes between brain endothelial cells with the idea to define suitable targets for manipulation of BBB junctions, the 3rd WP investigated transcription factors regulating the BBB cell-cell junctions. Three additional WPs focused on testing and verifying the strategies identified in the other WPs for paracellular delivery of large drugs across the BBB in vitro and in vivo in health and disease. Finally, two WPs were dedicated to the successful management and networking and dissemination of results within and beyond JUSTBRAIN.
Besides the establishment for numerous novel approaches that can be explored in the future for opening BBB TJs for the delivery of large drugs into the CNS, the JUSTBRAIN collaborative research has achieved its major goal by identifying with the canonical Wnt/-catenin signaling pathway one molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large drugs into the brain in vivo during health and neuroinflammation.
JUSTBRAIN has therefore achieved breakthroughs in the development of paracellular bypasses of the BBB for the safe delivery of large drugs into the brain.
Project Context and Objectives:
There is a lack of therapies for most neurological disorders ranging from rare, however, fatal diseases such as lysosomal storage diseases and primary brain tumors to major public health concerns such as brain metastases, multiple sclerosis, stroke or Alzheimer’s disease. In addition to the development of effective drugs, the delivery of diagnostic or therapeutic compounds into the brain has become a major challenge in successfully treating these diseases. To maintain homeostasis of the brain, passage of potentially harmful substances from the blood stream into the brain is restricted by the highly specialized vascular barrier, the blood-brain barrier (BBB). In this neuroprotective role, the BBB also hinders the delivery of many potentially important diagnostic and therapeutic drugs to the central nervous system (CNS). Current estimates are that only 2% of small molecule drugs and negligible amounts of large molecule drugs in clinical use cross the BBB. This has dramatically slowed the development of pharmacotherapies and immunotherapies in brain diseases. Because most drugs cannot penetrate the BBB, the treatment of diseases as primary brain tumors or brain metastases is presently reduced to symptomatic and palliative measures.
JUSTBRAIN research focus
The BBB inhibits the free trans-cellular passage of hydrophilic molecules by an extremely low pinocytotic activity and their paracellular diffusion by an elaborate network of complex TJs between its endothelial cells. Specific transport or receptor-mediated transcytosis systems, selectively expressed by brain endothelial cells to ensure the effective delivery of nutrients from the blood to the brain and the rapid removal of toxic metabolites from the brain have been exploited for drug delivery. Unspecific opening of the endothelial junctions as performed in selected clinics today, i.e. by hyperosmotic shock, bears a high risk of toxicity as it is accompanied by the influx of substances other than the drug of interest into the brain and may thus culminate in global adverse drug reactions. Therefore, controlled and transient opening of BBB TJs is required for safe paracellular drug delivery into the brain.
JUSTBRAIN was dedicated to specifically explore and develop strategies targeting individual junctional proteins for a controlled and transient – and thus safe - opening of BBB junctions allowing for the delivery of large drugs into the brain. This approach was considered particularly difficult so far due to the poor knowledge of BBB junction organization and, most importantly, for the lack of treatments able to target these structures specifically and reversibly. Prior to the beginning of their collaboration, the groups of the JUSTBRAIN consortium had collected a large set of data during their previous work, which offered a unique platform for the development of novel and targeted therapeutic approaches to manipulate paracellular BBB permeability. In order to proceed in a fast and competitive way, these groups combined their complementary approaches, which included in vitro and in vivo tests and animal models of CNS pathologies, (e.g animal models for multiple sclerosis) where BBB opening may be therapeutic. The wide range of expertise offered by this consortium was unique in Europe for the particular approach of targeting BBB junctions for paracellular drug delivery into the brain.
In pursuit of this goal JUSTBRAIN aimed to identify an entirely novel platform of drugable molecular targets to bypass the BBB and thereby improve delivery of large molecules into the brain and thus expand on diagnostic and therapeutic possibilities for neurological disorders.
To achieve this goal JUSTBRAIN
• Defined the best known molecular targets of BBB junctions (trans-membrane junctional adhesion proteins and/or their intracellular binding partners) by applying available inhibitors and therapies in health and disease
• Characterized novel junctional adhesion proteins and/or their intracellular binding partners in health and disease
• Characterized the transcriptional control of BBB junction maintenance in health and disease.
• Transiently increased or decreased expression of other/novel junctional proteins (JAMs, L1) at the BBB in vitro and in vivo
• Modulated the extracellular homophilic or heterophilic interaction of tight junction proteins, i.e. claudins at the BBB in vitro and in vivo
• Choose feasible approaches for paracellular drug delivery to be further developed for potential clinical development
Project Results:
JUSTBRAIN has made an in depth analysis of the molecular composition of the cell-to-cell contact complexes in between brain microvascular endothelial cells establishing adherens junctions (AJs) and tight junctions (TJs) at the blood-brain barrier (BBB) in vitro and in vivo. In addition it characterized the presence of molecules localized in cell-to-cell contacts of the BBB outside of the organized junctions.
Analysis was performed in mouse models investigating healthy mice and mouse models for Alzheimer’s disease, multiple sclerosis, brain tumors and vascular malformations modelling cerebral cavernous malformations. Additional analysis was performed in a number of different mouse and human in vitro models for the BBB. In this context JUSTBRAIN specifically
• Described the molecular organization of AJs and TJs in endothelial cells in primary cultures from mouse brain and in mouse and human endothelial cell lines of brain origin
• Established culture conditions to induce stabilization of AJs and TJs in brain endothelial cells in culture
• Confirmed the composition of endothelial junctions in brain samples and in retinae from mice, transgenic and pathological models
• Identified one cytoplasmic component of AJs as crucial for the maintenance of barrier properties and for the organization of tight junctions and established a BBB transcript signature that defines BBB TJs.
Furthermore JUSTBRAIN established novel transgenic mouse models for mimicking human diseases affecting the BBB and allowing for deletion or increased expression of specific molecules localized in BBB AJs (CCM1, -2 and -3) and TJs and cell-to-cell contacts.
Using these models JUSTBRAIN identified transmembrane components of AJs and TJs that are crucial for enhancing the barrier properties of brain endothelial cells.
Cerebral cavernous malformations (CCM) are vascular malformations of the (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. CCM lesions are formed by enlarged and irregular blood vessels. In addition, as the CCM complex can associate with adherens junctions an important role in junctional integrity of the endothelium is suggested. Indeed loss of function of any CCM protein strongly affects AJ organization and AJ permeability. Most importantly, vascular fragility is markedly increased, resulting in lesions that frequently bleed and cause headache, seizures and eventually hemorrhagic stroke in the affected patients.
JUSTBRAIN therefore established by deleting CCM1, or -2 or -3 mouse models for CCM as a tool to identify signaling pathways governing adherens and tight junctions in endothelial cells of the brain. JUSTBRAIN showed that postnatal deletion of any of the three CCM genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. We showed that endothelial-specific disruption of the CCM1 gene in mice induces Endothelial-to-mesenchymal transition (EndMT), which contributes to the development of vascular malformations. CCM-1-ablated endothelial cells showed upregulation of endogenous BMP6 which activated the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway leading to EndMT. We could demonstrate that inhibition of TGF-β and BMP singnaling pathways prevented EndMT both in vitro and in vivo and reduced the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. In further studies, we have observed that the first event in the induction of the EndMT phenotype following loss of CCM3 gene is the activation of Wnt/-catenin signalling. This is then followed by activation of the TGF-/BMP. Pharmacological inhibition of -catenin transcriptional signalling inhibited the progression of vascular malformations in vivo. Therefore, a cross-talk exist between two crucial signalling pathways, that both regulate terminal differentiation in several cellular context.
CCM is one of the first conditions described that shows that altered junctional composition of BBB cell-to-cell junctional organization can affect vascular morphogenesis and can be the cause of severe pathologies. Thus, exploration of the CCM complex and signaling pathways in JUSTBRAIN established novel insight on a molecular complex critically involved in controlling junctional integrity and hence on the future possibility for targeting of BBB junctional complexes for therapeutic opening of BBB TJs for drug delivery.
On the other hand JUSTBRAIN accumulated a vast amount of “negative” data demonstrating that although expressed in BBB cell-to-cell junctions numerous molecules can be deleted without loss of function for BBB junctional integrity. Examples include the family of junctional adhesion molecules (JAMs), which within JUSTBRAIN were identified to not provide suitable target molecules for transient opening of BBB TJs. JUSTBRAIN has established that, unlike in other vascular barriers, the JAM family is not critically involved in regulating the integrity of BBB TJs and AJs in vivo and in vitro. Specifically, mouse models with deletions for JAM-A, JAM-B or JAM-C and endothelial overexpression of JAM-C did not result in loss of barrier functions at the BBB in vitro and in vivo. At the same time JAMs do influence leukocyte trafficking across the BBB. Therefore JAM-A, JAM-B and JAM-C are unsuitable targets for regulating BBB TJs and AJs. Although this is a negative finding it emphasizes that BBB TJs are different from TJ in other vascular barriers, which might allow for BBB-specific manipulations of TJ integrity.
Further molecules that could be excluded as targets for manipulating BBB TJ integrity to allow for the delivery of large molecular drugs across the BBB were: ICAM-1, CD73 and L1.
Using mouse models with increased or deleted expression of claudins (Clds), thus altering BBB TJ composition, JUSTBRAIN established first of all that Cld-1 is not expressed at the BBB. However, sealing of BBB TJs by ectopic expression of Cld-1 in BBB endothelium reduces BBB leakiness and ameliorates clinical disease in an animal model for multiple sclerosis, thus showing that modulation of Cld localization in BBB TJs is critical for controlling BBB TJ permeability. Using our in vitro model of human BBB, the hCMEC/D3 cell line, we identified a new partner of Cld5 that regulates TJ integrity: the Guanine nucleotide binding protein Gi2 (Luissint et al. JCBFM 2012).
At the beginning of JUSTBRAIN one of the most promising strategies for transient opening of the BBB TJs was therefore considered to be the modulation of TJ integrity by modulatory targeting of the homophilic or heterophilic interactions of claudins in cis and trans by the use of Cld-targeting peptides. JUSTBRAIN demonstrated for the first time that the first extracellular loops (ECL1) of Cld1 and Cld5 are involved in homo- and heterophilic interactions between classic Clds. The interactions were shown to be redox-sensitive, which is of relevance for pathological conditions related to oxidative stress.
Inhibition of these homo- and heterophilic Cld-interactions therefore seemed a promising approach for the transient opening of BBB TJs for the delivery of large drugs across the BBB. To this end, we developed Cld-targeting peptides based on the amino acid sequences of the ECLs of a number of Clds and characterized and tested their ability to transiently open BBB TJs. Cld-targeting peptides related to the first extracellular loop of Cld1 (especially peptides of the C-terminal half of Cld1-ECL1, e.g. mC1C2) were found to increase paracellular permeability of in vitro mouse models of the BBB in a concentration- and time-dependent manner without exerting toxic effects on BBB mouse endothelial cells in culture. However, it has to be mentioned that these peptides were found to be toxic to the human brain endothelial cell line hCMEC/D3. The most significant results were obtained with mC1C2. Modifications at the C-terminus may even improve the BBB TJ opening properties of mC1C2. mC1C2 was found to bind to Cld1 and to Cld5 with nanomolar binding constants explaining how it can target BBB endothelium lacking Cld1 expression but showing high expression of Cld5. The mechanism of action defined was that mC1C2 internalized Cld1 via the clathrin pathway which, in turn, caused delocalization of classic Clds from the membrane to cytosol by a mechanism not fully understood. In general, mC1C2 therefore seems to reversibly modulate tissue barriers via specific and unspecific binding to the ECL1 of classic Clds, inducing down-regulation of membranous Clds.
Unfortunately, this approach completely failed in vivo where JUSTBRAIN could demonstrate that systemic injection of Cld-targeting peptides that allowed for transient opening of BBB TJs in vitro exerts toxic effects leading to sudden death and induction of seizures in injected animals. Respecting the 3R rules of animal experimentation JUSTBRAIN decided that more in depth in vitro research is required to fully understand the mode of action of these peptides before aiming for a detailed analysis of their potential for transient opening of BBB TJs in vivo.
Thus JUSTBRAIN could exclude numerous approaches, e.g. targeting the junctional adhesion molecules or claudins with Cld-targeting peptides either due to the lack of a specific function of these molecules on modulating paracellular permeability at the BBB or due to severe toxic side effects exerted by these approaches.
However, an alternative approach might be the targeting of Clds expressed at the BBB by monoclonal antibodies specifically recognizing the extracellular domains of Clds. Such antibodies have been developed in JUSTBRAIN and might provide a safer alternative for interrupting the homophilic and heterophilic Cld interactions in cis and trans for the transient opening of BBB TJs.
When investigating the role of Wnt/β-catenin signalling in maintaining integrity of BBB TJs we discovered that the non-canonical Wnt/PCP pathway is also involved in the regulation of BBB TJ integrity and manipulation of this signalling pathway translates in opening of BBB TJs. Specifically, we showed that inhibition of the non-canonical Wnt/PCP pathway by knocking-down PAR3, a component of the PAR3/PAR6/PKC complex, increased the permeability of in vitro models of the BBB. Interestingly, this alteration in permeability was limited to small compounds (LY and 4kDa-dextran) while no change of the BBB permeability for high MW compounds (70kDa- Dextran and FITC-GAM Antibody) could be observed. Thus, targeting the non-canonical Wnt/PCP pathway, discovered in JUSTBRAIN to modulate BBB TJs, did allow the reproducible opening of the BBB for the passage of small but not large molecules across the BBB. These findings are potentially of clinical importance, suggesting that controlling this pathway might be considered at least as an adjunct strategy for the regulation of BBB permeability.
Excluding on the one hand numerous approaches for the therapeutic manipulation of BBB TJs in vivo, JUSTBRAIN reached its overall goal, which was to define at least one molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large drugs into the brain in vivo during health and neuroinflammation.
The canonical Wnt/-catenin signalling pathway was shown to be relevant for BBB formation and BBB maintenance in the mouse in vivo and in vitro.
Specifically JUSTBRAIN demonstrated that
• the Wnt/-catenin signalling pathway is also involved in the maturation of a human in vitro BBB model – hCMEC/D3 - by inducing the expression of claudin-3 and probably claudin-5
• catenin transcription is required for BBB maintenance in brain ECs in vivo in transgenic mouse models of catenin gain-of-function and loss-of-function mutations thus demonstrating that manipulating Wnt/β-catenin signalling in vivo in transgenic mouse models allowed to regulate the permeability of BBB TJs
• Wnt1, but not control- or Dkk1- expressing GL261 glioma cells showed maintained barrier characteristics of tumour vessels in vivo.
• activation of catenin transcription by Wnt3a as well as by LiCl or BIO increased barrier properties and expression of TJ genes (Cld-3 and -5) in mouse and human in vitro models of the BBB.
• By identifying novel binding partners of AJ (e.g. CCM1, CCM3) and regulators of the Wnt/-catenin signalling pathway controlling BBB junctional integrity that this pathway can be even further explored for safe pharmacological targeting with the goal to open BBB TJs for drug delivery.
• that barrier properties of in vitro BBB models can be improved by stimulating the Wnt/β-catenin signalling, allowing for the establishment of novel and easy to handle in vitro BBB models for high throughput screening
• that small molecular compounds can be used to pharmacologically inhibit the transcriptional activity of catenin in brain ECs in order to transiently open the BBB tight junctions in vitro and in vivo
Thus JUSTBRAIN has identified the canonical Wnt/-catenin signalling pathway as the most promising molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs for the delivery of large molecular drugs into the brain in vivo during health and neuroinflammation.
Potential Impact:
By discovering the canonical Wnt/-catenin signalling pathway as a molecular pathway that can be pharmacologically targeted for the manipulation of BBB TJs and potentially of the metabolic BBB, allowing for the delivery of large molecular drugs into the brain in vivo during health and neuroinflammation, JUSTBRAIN has established a breakthrough in the development of paracellular bypasses of the BBB for the safe delivery of large molecular drugs into the brain.
The major impacts of JUSTRBRAIN are:
Scientific and clinical impacts
• The demonstration of proof-of-principle of a novel therapeutic strategy for drug delivery across the BBB, which may be developed into future clinical applications.
• A common set of research tools and technologies, which may lead to therapeutic applications in many different pathological conditions.
• The possibility to increase availability of effective medical products to CNS. Many effective drugs showed limited success in the clinic due to their limited capacity to effectively cross BBB.
• Generate a more profound understanding of the biology of the BBB permitting a better design of therapeutic strategies through the cell biology to pre-clinical feedback loop applied. Most products are still in early stage of development due to the absence of easy, low cost, clear-cut assays for BBB penetrance.
• Identify approaches to generate high value and high impacting therapeutic agents, which European companies can effectively exploit.
European Strategic Impacts
• The development of a high-value new technology for the delivery of large drugs into the brain
• To boost the European biotechnology industry, and contribute to developing standard protocols to test drugs for crossing the BBB
• Improve competitiveness of European pharmaceutical companies with highly innovative products that will permit European companies to become global players.
Socio economic impacts
• The development of an European capability in diagnosis and therapy of the increasing number of neurological disorders in an ageing population, which are currently either untreatable or with insufficient therapy (brain tumors, multiple sclerosis, stroke, Alzheimer's Dementia etc.) with the potential for combating rising health costs.
• Facilitate the creation of novel therapeutics, which will decrease the burden for European citizens and health agencies of neurodegenerative diseases.
List of Websites:
Project website: www.JUSTBRAIN-fp7.eu
Coordinator contact: Prof. Britta Engelhardt, Theodor Kocher Institut Bern, University of Bern, Switzerland, E-Mail: bengel@tki.unibe.ch