Yosuke Hashimoto

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key …

実行可能なのではないかと考えるようになった. しかし, CNS 疾患を誘発するような CLDN-5 のアミノ酸変異は, これまで報告されていなかった. そのため, 欧州のさまざまな病院と連携し, CLDN-5 の遺伝子型を調査していった結果, 2 名の小児交互性片麻痺 (Alternating Hemiplegia of Childhood, AHC) の患者から, CLDN-5 の細胞外領域に位置する 60 番目のアミノ酸のグリシンがアルギニンに置換された変異体が発見された 4). 解析の結果, CLDN-5 G60R 変異体は, 野生型の CLDN-5 が有する低分子に対するバリア形成能力が著しく低いのみだけでなく, アニオンの透過性が著しく高いイオンチャネルのようなバリアを形成する能力を獲得していることがわかった. G60 が存在する部位は, CLDN-15 がカチオンチャネルとして機能するために必須の負電荷のアスパラギン酸が存在する部位の近傍である. そのため, この部位に正電荷のアルギニンが存在することにより, バリア形成型の CLDN-5 が CLDN-15 のようなイオンチャネル形成型の CLDN に変異したことが推察される. すなわち, この CLDN-5 G60R 変異を有する患者は, アニオンの透過性が著しく高い BBB により, 脳内のイオンバランスが崩壊しているため, AHC を発症していることが強く疑われる. AHC を引き起こす遺伝子変異は神経細胞に発現する ATP1A3 遺伝子 (Na+/K+transporter) の変異のみが報告されており, AHC は channelopathy だと考えられている. CLDN-5 G60R 変異体, もしくは, 同様のチャネル形成能を獲得するような CLDN-5 の変異は, AHC や AHC に似た疾患である片麻痺性片頭痛の原因となる可能性が非常に高い. 現在はモデルマウスを開発中であり, このマウスを用いてより詳細な AHC 発症メカニズムの解析と, G60R 変異体に特異的な抗 CLDN-5 抗体を用いた治療実験を行っていく予定である.

The CLDN5 gene encodes claudin-5 (CLDN-5) that is expressed in endothelial cells and forms tight junctions which limit the passive diffusions of ions and solutes. The blood–brain barrier (BBB), composed of brain microvascular endothelial cells and associated pericytes and end-feet of astrocytes, is a physical and biological barrier to maintain the brain microenvironment. The expression of CLDN-5 is tightly regulated in the BBB by other junctional proteins in endothelial cells and by supports from pericytes and astrocytes. The most recent literature clearly shows a compromised BBB with a decline in CLDN-5 expression increasing the risks of developing neuropsychiatric disorders, epilepsy, brain calcification and dementia. The purpose of this review is to summarize the known diseases associated with CLDN-5 expression and function. In the first part of this review, we highlight the recent understanding of how other …

We thank our colleagues Panagiotakaki et al. 1 for their interest in our recent work elucidating the functional relevance of the G60R mutation in the CLDN5 gene in two unrelated cases of children presenting with alternating hemiplegia. Our study represents the first report of a dominant acting mutation in the coding sequence of the CLDN5 gene and has, we believe, major implications for our understanding of blood–brain barrier (BBB) function and tight junction biology in general. 2 In reference to queries from patients and patient groups, we would posit that mutations in CLDN5 are most certainly not going to be a leading cause of alternating hemiplegia of childhood (AHC). However, while further research on BBB disruption and indeed the possibility of CLDN5 variants being causative of hemiplegia is warranted, it would not be an arduous exercise to include CLDN5 screening in any case of suspected hemiplegia or …

Claudin-5 is the most enriched tight junction protein at the blood–brain barrier. Perturbations in its levels of expression have been observed across numerous neurological and neuropsychiatric conditions; however, pathogenic variants in the coding sequence of the gene have never been reported previously. Here, we report the identification of a novel de novo mutation (c.178G>A) in the CLDN5 gene in two unrelated cases of alternating hemiplegia with microcephaly. This mutation (G60R) lies within the first extracellular loop of claudin-5 and based on protein modelling and sequence alignment, we predicted it would modify claudin-5 to become an anion-selective junctional component as opposed to a purely barrier-forming protein. Generation of stably transfected cell lines expressing wild-type or G60R claudin-5 showed that the tight junctions could still form in the presence of the G60R mutation but that …

Claudin-5 is the dominant tight junction protein in brain endothelial cells and exclusively limits the paracellular permeability of molecules larger than 400 Da across the blood–brain barrier (BBB). Its pathological impairment or sustained down-regulation has been shown to lead to the progression of psychiatric and neurological disorders, whereas its expression under physiological conditions prevents the passage of drugs across the BBB. While claudin-5 enhancers could potentially act as vascular stabilizers to treat neurological diseases, claudin-5 inhibitors could function as delivery systems to enhance the brain uptake of hydrophilic small-molecular-weight drugs. Therefore, the effects of claudin-5 manipulation on modulating the BBB in different neurological diseases requires further examination. To manipulate claudin-5 expression levels and function, several claudin-5 modulating molecules have been developed. In this review, we first describe the molecular, cellular and pathological aspects of claudin-5 to highlight the mechanisms of claudin-5 enhancers/inhibitors. We then discuss recently developed claudin-5 enhancers/inhibitors and new methods to discover these molecules.

Claudin-5 (CLDN-5) is an essential component of the tight junction seal in the blood–brain barrier. Previously, we showed that CLDN-5 modulation in vitro via an anti-CLDN-5 monoclonal antibody (mAb) may be useful for increasing the permeability of the blood–brain barrier for drug delivery to the brain. Based on these findings, here we examined the safety and efficacy of the anti-CLDN-5 mAb in a non-human primate. Cynomolgus monkeys were intravenously administered the anti-CLDN-5 mAb followed by fluorescein dye (376 Da), and the concentrations of the dye in the cerebrospinal fluid was examined. When the mAb was administered at 3.0 mg/kg, the concentration of dye in the cerebrospinal fluid was increased, and no behavioral changes or changes in plasma biomarkers for inflammation or liver or kidney injury were observed. However, a monkey that received the mAb at 6 mg/kg experienced convulsions …

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HighlightsModulation of tight junctions may overcome the blood–brain barrier (BBB).Claudin-5 is the main component of BBB tight junctions.The composition of BBB tight junctions is not fully known.The ROCK and VE-cadherin/β-catenin pathways are targets for BBB modulation.RNAi agents and direct-binding molecules may enable size-selective BBB passage.Overcoming the blood–brain barrier (BBB) to enable the treatment of central nervous system (CNS) diseases is an active field of research. Modulating or opening the tight junctions (TJs) in brain endothelial cells is one method to enable a range of small-molecular-weight drugs to cross the BBB via the paracellular route. Over the past 2 decades, the molecular understanding of TJ proteins in the BBB has significantly improved, and several agonists and antagonists have been tested for modulation of the TJs. In this review, we discuss the composition of TJ …

The blood-brain barrier (BBB) is the one of the most robust physical barriers in the body, comprised of tight junction (TJ) proteins in brain microvascular endothelial cells. The need for drugs to treat central nervous systems diseases is ever increasing, however the presence of the BBB significantly hampers the uptake of drugs into the brain. To overcome or circumvent the barrier, many kinds of techniques are being developed. Modulating the paracellular route by disruption of the TJ complex has been proposed as a potential drug delivery system to treat brain diseases, however, it has several limitations and is still in a developmental stage. However, recent significant advance in medical equipment /tools such as targeted ultra-sound technologies may resolve these limitations. In this review, we introduce recent advances in site- or molecular size-selective BBB disruption/modulation technologies and we include …