Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood–brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells. TOR (target of rapamycin) was originally identified in two mutantSaccharomyces cerevisiae strains, TOR1-1 and TOR2-1, that are resistant to the growth-inhibiting effect of the immunophilin-immunosuppressant complex FKBP (FK506 binding protein) and rapamycin (17). TOR1 and TOR2 are large proteins (Ϸ280 kDa) and are Ϸ70% identical (26,28 (21, 48). mTOR and other members of this family, including ATM, ATR/FPR, and DNA-PKcs, contain C-terminal regions with high similarity to the catalytic domains of phosphoinositide (PI)-3 kinase and PI-4 kinase (26, 28). However, PIKK members are not lipid kinases but rather function as serine-threonine kinases (4, 20). The PIKK proteins contain a short segment at the extreme C terminus that is essential for protein kinase activity and is not present in PI-3 and PI-4 kinases (51).Cell culture studies have demonstrated that mTOR controls protein synthesis, in part by phosphorylating downstream substrates, including p70 s6 kinase (p70 S6K1 ) (3, 5, 20) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) (4, 5, 13, 15). p70 S6K phosphorylates the 40S ribosomal protein S6 and is proposed to play a crucial role in the translation of 5Ј-terminal oligopyrimidine tract mRNAs, which primarily encode ribosomal proteins and components of the translation apparatus (22, 23). Phosphorylation of 4E-BP1 disrupts its binding to eIF4E, a protein that binds the 5Ј cap structure of mRNA. Released eIF4E then forms a functional translation initiation complex with eIF4G, eIF4A, and eIF3 ribosomes, enhancing translation (29, 45). Inactivation of 4E-BP1 and family proteins has a profound effect on translation of mRNAs with complex 5Ј untranslated regions, which often encode regulatory proteins such as protooncogenes (45). The recent discoveries of a 150-kDa binding partner of mTOR, named raptor (regulatory-associated protein of mTOR) (14,27), and its Saccharomyces cerevisi...

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.

We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.