[R01] Structure-Guiding Tools to Study the Role of Choline in the Development of Congenital Hydrocephalus
Ente: National Institute of Neurological Disorders and Stroke
Scadenza: 2030-07-31
Importo max: 629.173 EUR
Paese: US
Descrizione
PROJECT SUMMARY
The blood-brain barrier (BBB) is a term used to describe the unique properties of the blood vessels that
vascularize the central nervous system (CNS) and restrict the movement of nutrients, ions and cells between
the blood and the brain. BBB dysfunction contributes to various CNS diseases and greatly impedes drug delivery.
Despite its importance, little progress has been made in manipulating the BBB due to a major gap in our
understanding, and a lack of tools to interrogate, constituents of the BBB at a molecular and cellular level. We
have found that mutations in the blood brain barrier (BBB) choline transporter, FLVCR2, or the related choline
and ethanolamine transporter, FLVCR1 (expressed in brain neuroprogenitor cells, NPCs), cause congenital
hydrocephalus (CH). However, the mechanisms by which FLVCR1 and FLVCR2 transport choline and contribute
to the development of CH are unknown, and we currently lack the tools to probe these mechanisms at a
molecular level. In this proposal we will address this void through three specific Aims: In Aim 1, we will
determine the structures of FLVCR1 and FLVCR2 using cryo-electron microscopy (cryoEM), so as to provide a
model to generate structure-based functional hypotheses. In Aim 2, using radioactive and fluorescently tagged
choline tracers and innovative heme reporters, combined cell-culture techniques, we will define the mechanisms
of choline transport and heme-choline interactions in brain endothelial cells and NPCs, and test structure-based
hypotheses in vitro. In Aim 3, we will utilize CRISPR/CAS9 to generate a series of new mouse models of CH
bearing conditional mutation in FLVCR1 or FLVCR2 to test structure:function relationships in vivo. These new
tools will be freely disseminated to the scientific community and should have a catalytic effect on the field. Our
preliminary data include cryoEM analysis of recombinant FLVCR2 and complex formation with FLVCR1/2-cross
reactive antibodies (Fabs); the generation of CRISPR/CAS9-mediated Flvcr2 knockout brain endothelial cells,
Flvcr1 knockout HEK293 cells, and a NPC culture platform; the generation of FLVCR2 brain endothelial cell
specific conditional knockout/knockin and FLVCR1 NPC specific conditional knockout mouse models of CH.
These data demonstrate that we are well-poised for structural determination and the development of an
adaptable cell-based assays and genetic mouse models for future structure/function studies on FLVCR1 and
FLVCR2, and their collaborative roles in BBB choline transport and CH.
Istituzione: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
PI: Thomas Darmody Arnold
Progetto: 5R01NS142590-02
Settori: National Institute of Neurological Disorders and Stroke
Vai al bando originale
Registrati gratis su Bandolo per trovare bandi compatibili con la tua azienda.