Research
Establishment of central nervous system barrier-based drug discovery science to open up the future of central nervous system disease therapy
In addition to the blood-brain barrier (BBB), there are three other cellular barriers in the central nervous system (CNS): the blood-cerebrospinal fluid barrier (BCSFB), the blood-arachnoid barrier (BAB) and the blood-spinal cord barrier (BSCB), which separate the peripheral blood circulation and CNS tissues. We call these 4 barriers as the “CNS barriers”
Challenges in the development of new drugs for the treatment of CNS diseases
(1) The general concept is that the molecular targets of drugs to treat CNS diseases are expressed in the CNS tissues, not in the CNS barriers. (2) Thus, drugs need to pass through the CNS barriers to reach the CNS tissues, but more than 99% of drugs are prevented from entering the CNS tissues by efflux pumps, such as P-glycoprotein (P-gp), in the CNS barriers. (3) Proteins, genes, nanoparticles and other macromolecules cannot pass through the CNS barrier at all. Thus, we cannot expect any positive future in which we can efficiently develop drugs for the treatment of CNS diseases without innovative technologies and ideas.
To open up a new drug discovery field for CNS diseases, we focus on the cells in the CNS barriers themselves as a target for therapeutic and diagnostic drugs to solve the problem regarding poor drug uptake into CNS tissues (as barrier-targeting drugs do not need to penetrate the CNS barriers). Since the CNS barriers maintain the brain environment and homeostasis, the abnormality of the CNS barriers is a cause of CNS diseases (although the barrier also gets abnormal as a result of the progression of the CNS diseases). Our goal is to establish a new and unprecedented drug discovery strategy to treat and diagnose CNS diseases by treating and diagnosing the abnormalities of the barriers. We are working hard to realize the following four concepts, with the goal of creating a new academic discipline “CNS barrier-based Drug Discovery Science”.
Concept 1 We have elucidated the pathomechanisms of the CNS barriers in CNS diseases and prove their contribution to the onset and progression of CNS diseases. Our strength is that we possess some high-resolution mass spectrometers for quantitative proteomics to elucidate the molecular pathomechanisms of the CNS not only in mice but also in humans. In particular, acquiring quantitative and comprehensive proteomics data is difficult in human tissues that are usually stored in formalin. We have developed our own highly accurate quantitative proteomics technology for formalin-fixed paraffin-embedded (FFPE) sections. We are performing quantitative proteomic analysis of the human CNS barriers with CNS diseases in FFPE sections using this technology coupled with laser microdissection. We will prove the contribution of the responsible molecules or molecular mechanisms identified by quantitative proteomics analysis to the onset and progression of CNS diseases by regulating their expression in a cell-type specific manner in the CNS tissues using adeno-associated virus (AAV) system.
Concept 2 We will establish a technology to efficiently deliver antibodies into the inside of cells in the CNS barriers by identifying membrane proteins that are more highly expressed in the CNS barriers than in other organs. Our strength is that we have developed a technology to comprehensively and precisely quantify the absolute expression levels (mole) of membrane proteins (quantitative Global Absolute Proteomics (qGAP) method). Using this technology, we can comprehensively elucidate the absolute expression levels of membrane proteins localized on the blood-side plasma membrane of CNS barrier cells. Then, we can select membrane proteins that are selectively and highly expressed in each CNS barrier compared to other organs. We will generate antibodies against the extracellular regions of the candidate membrane proteins, and select the antibodies that are rapidly taken up into the CNS barrier cells from a number of antibody candidates.
Concept 3 We will demonstrate the therapeutic efficacy of our CNS barriers-targeing approaches for CNS diseases. Our strength is that we have developed own algorithms that can predict suitable small molecule drugs or nucleic acid sequences (miRNA, ASO, siRNA) to normalize the diseases-related abnormal protein-expression profiles. For example, conventional methods to predict therapeutic miRNA sequences select suitable sequences to normalize the diseases-related abnormal mRNA-expression profiles based on transcriptome data and miRNA-binding sites. However, as not all mRNA-expression levels correlate well with protein-expression levels, they are unable to sufficiently normalize the diseases-related changes. In contrast, the “miR-PAIR method”, an algorithm we developed, can predict miRNA sequences that can normalize the diseases-related abnormal protein-expression profiles directly based on proteomics data and our unique network analysis. Using these techniques, we will select small molecule drugs, miRNAs, and ASO/siRNAs that can normalize the molecular pathomechanisms of the CNS barriers to be elucidated in Concept 1 (nucleic acids will be conjugated with antibodies in Concept 2), and demonstrate that they can treat CNS disease pathologies using animal models of CNS diseases.
Concept 4 We will establish the diagnostic method to evaluate the normality of CNS tissues (or the prograssion of CNS diseases) by measuring the state of the CNS barriers. By conjugating an imaging probe to the antibody to be developed in Concept 2 and administering it, a diagnostic probe can be accumulated/internalized into the CNS barrier cells and the environment of the CNS barriers can be measured. Since the CNS tissues and the CNS barriers interact with each other, abnormalities in the CNS tissues will appear in the CNS barriers as well. In addition to imaging probe-based diagnosis, we also develop the blood-based diagnosis for disease biomarkers that leak from the CNS barrier cells into the bloodstream, in order to establish a new diagnostic foundation “CNS barrier diagnosis” for CNS diseases.
Hashimoto‘s Group (Claudin-5 study)
The research interest of Hashimoto’s group is Claudin-5 (CLDN-5) protein in the blood-brain barrier, which is one of the four CNS barriers proposed by Uchida’s group. Claudin-5 is the responsible protein to build tight junctions in brain microvascular endothelial cells, the major cellular component of the blood-brain barrier.
Tight junctions are developed after epithelial or endothelial cells develop adherens junctions in the paracellular clefts. The two adjacent plasma membranes are fused in the tight junctions to limit the paracellular diffusion of solutes and ions. Tight junctions are formed by the CLDN family that has 26 members in human. Each CLDN has unique tight junction-forming or -modulating abilities with different cell-type/tissue expression profiles. For examples, a monolayer of brain microvascular endothelial cells, where tight junctions are mainly composed of CLDN-5, do not allow paracellular diffusions of either solutes or ions; while, that of small intestinal epithelial cells, where tight junctions contain CLDN-15, show higher paracellular cation permeability. When the tight junctions are disrupted, a wide-range of diseases are induced due to the disrupted tissue homeostasis. When CLDN-5 expression/function is impaired, following neurological symptoms are mainly induced: seizure, cognitive decline and depression/anxiety. Added to this, the impairments in CLDN-5 expression/function increase the severity and chronicity of many neurological disorders. Dr Yosuke Hashimoto who leads this research group is a specialist in studies relating to CLDN-5. He has discovered a novel pathogenic CLDN-5 missense mutatnt (https://doi.org/10.1093/brain/awac215) and has generated novel monoclonal antibodies against CLDN-5 that can neutralize CLDN-5 function (https://doi.org/10.1124/jpet.117.243014).
The mission of Hashimoto’s group is to cure CNS diseases caused by the abnormal CLDN-5 expression/function via its normalization. We are challenging to
- Neutralize a pathogenic CLDN-5 mutant selectively in the body.
- Inhibit genes that negatively regulate CLDN-5 expression under the CNS diseases.
We have used following research tools to achieve our mission. We have many collaborators regardless of nationality and many of these tools/techniques have been imported from these collaborators.
- Genetically engineered mice (mouse models for Cldn5-mediated neurological disease, Cldn5 promoter-driven GFP-expressing transgenic mice)
- Genetically engineered cellular models (virus-mediated gene transduction, CRISPR/Cas9)
- Antibody (generation of monoclonal antibodies using immunization, antibody-engineering, and targeting to the blood-brain barrier)
- Microphysiological systems for recapitulating the blood-brain barrier (the techniques will be imported from overseas)