Identification of ATP8B1 as a blood-brain barrier-enriched protein.

In order to define the molecular anatomy of the blood-brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.

Lipopolysaccharide alters the blood-brain barrier transport of amyloid beta protein: a mechanism for inflammation in the progression of Alzheimer's disease.

Alzheimer's disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone.

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys --> Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.

Carbonic anhydrase XIV in skeletal muscle: subcellular localization and function from wild-type and knockout mice.

The expression of carbonic anhydrase (CA) XIV was investigated in mouse skeletal muscles. Sarcoplasmic reticulum (SR) and sarcolemmal (SL) membrane fractions were isolated from wild-type (WT) and CA XIV knockout (KO) mice. The CA XIV protein of 54 kDa was present in SR and SL membrane fractions as shown by Western blot analysis. CA activity measurements of WT and KO membrane fractions showed that CA XIV accounts for approximately 50% and 66% of the total CA activities determined in the SR and SL fractions, respectively. This indicates the presence of at least one other membrane-associated CA isoform in these membranes, e.g., CA IV, CA IX, or CA XII. Muscle fibers of the extensor digitorum longus (EDL) muscle were immunostained with anti-CA XIV/FITC and anti-sarco(endo)plasmic reticulum Ca(2+)-ATPase 1/TRITC, with anti-CA XIV/FITC and anti-ryanodine receptor/TRITC, or with anti-CA XIV/FITC and anti-monocarboxylate transporter-4/TRITC. CA XIV was expressed in the plasma membrane and in the longitudinal SR but not in the terminal SR. Isometric contraction measurements of single twitches and tetani and a fatigue protocol applied to fiber bundles of the fast-twitch EDL and of the slow-twitch soleus muscle from WT and KO mice showed that the lack of SR membrane-associated CA XIV did not affect maximum force, rise and relaxation times, and fatigue behavior. Thus, it is concluded that a reduction of the total SR CA activity by approximately 50% in CA XIV KO mice does not lead to an impairment of SR function.

Carbonic anhydrase II deficiency syndrome (osteopetrosis with renal tubular acidosis and brain calcification): novel mutations in CA2 identified by direct sequencing expand the opportunity for genotype-phenotype correlation.

The carbonic anhydrase II (CA II) deficiency syndrome is an autosomal recessive disorder that produces osteopetrosis, renal tubular acidosis, and cerebral calcification. Other features include developmental delay, short stature, cognitive defects, and a history of multiple fractures by adolescence. With one exception, all patients with osteopetrosis and renal tubular acidosis examined have proven to have CA II deficiency. All CA II-deficient patients analyzed have been found to have mutations in the CA2 gene. Previously, we used single strand conformational (SSCP) analysis to identify exons to be sequenced from CA II-deficient patients. In this report, we amplified all seven exons by PCR from genomic DNA and directly sequenced the amplified products. Application of this method allowed identification of eleven new mutations in 21 patients referred for confirmation of the diagnosis of CA II deficiency. These mutations were scattered over the genome from exon 2 to 7. In two opportunities for prenatal diagnosis, one from cultured amniocytes and one from chorionic villus biopsy, we demonstrated the general utility of the direct sequencing method for prenatal DNA diagnosis. These studies expand our knowledge of the heterogeneity in mutations underlying the CA II deficiency syndrome.

Expression, assay, and structure of the extracellular domain of murine carbonic anhydrase XIV: implications for selective inhibition of membrane-associated isozymes.

Carbonic anhydrase (CA) XIV is the most recently identified mammalian carbonic anhydrase isozyme, and its presence has been demonstrated in a number of tissues. Full-length CA XIV is a transmembrane protein composed of an extracellular catalytic domain, a single transmembrane helix, and a short intracellular polypeptide segment. The amino acid sequence identity of human CA XIV relative to the other membrane-associated isozymes (CA IV, CA IX, and CA XII) is 34-46%. We report here the expression and purification of both the full-length enzyme and a truncated, secretory form of murine CA XIV. Both forms of this isozyme are highly active, and both show an abrogation of activity in the presence of 0.2% SDS, in contrast to the behavior of murine CA IV. We also report the crystal structure of the extracellular domain of murine CA XIV at 2.8 A resolution and of an enzyme-acetazolamide complex at 2.9 A resolution. The structure shows a monomeric glycoprotein with a topology similar to that of other mammalian CA isozymes. Based on the x-ray crystallographic results, we compare and contrast known structures of membrane-associated CA isozymes to rationalize the structural elements responsible for the SDS resistance of CA IV and to discuss prospects for the design of selective inhibitors of membrane-associated CA isozymes.

Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney.

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO(3)(-) absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40-45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H(+)-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO(3)(-) absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.