PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

Activation of protein kinase C (PKC) has previously been shown to ameliorate the cholesterol transport defect in Niemann Pick Type C1 (NPC1) cells, presumably by increasing the soluble levels of one of its substrates, vimentin. This activity would then restore the vimentin cycle in these cells and allow vimentin-dependent retrograde transport to proceed. Here, we further investigate the effects of PKC activation in NPC1 cells by evaluating different isoforms for their ability to solubilize vimentin and correct the NPC1 cholesterol storage phenotype. We also examine the effects of PKC activators, including free fatty acids and the PKC-specific activator diazoxide, on the NPC1 disease phenotype. Our results indicate that PKC isoforms α, βII, and ε have the greatest effects on vimentin solubilization. Furthermore, expression or activation of PKCε in NPC1 cells dramatically reduces the amount of stored cholesterol and restores cholesterol transport out of endocytic vesicles. These results provide further support for the contribution of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.     

Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice

Preclinical studies of enzyme-replacement therapy for Fabry disease (deficient alpha-galactosidase A [alpha-Gal A] activity) were performed in alpha-Gal A-deficient mice. The pharmacokinetics and biodistributions were determined for four recombinant human alpha-Gal A glycoforms, which differed in sialic acid and mannose-6-phosphate content. The plasma half-lives of the glycoforms were approximately 2-5 min, with the more sialylated glycoforms circulating longer. After intravenous doses of 1 or 10 mg/kg body weight were administered, each glycoform was primarily recovered in the liver, with detectable activity in other tissues but not in the brain. Normal or greater activity levels were reconstituted in various tissues after repeated doses (10 mg/kg every other day for eight doses) of the highly sialylated AGA-1 glycoform; 4 d later, enzyme activity was retained in the liver and spleen at levels that were, respectively, 30% and 10% of that recovered 1 h postinjection. Importantly, the globotriaosylceramide (GL-3) substrate was depleted in various tissues and plasma in a dose-dependent manner. A single or repeated doses (every 48 h for eight doses) of AGA-1 at 0.3-10.0 mg/kg cleared hepatic GL-3, whereas higher doses were required for depletion of GL-3 in other tissues. After a single dose of 3 mg/kg, hepatic GL-3 was cleared for > or =4 wk, whereas cardiac and splenic GL-3 reaccumulated at 3 wk to approximately 30% and approximately 10% of pretreatment levels, respectively. Ultrastructural studies demonstrated reduced GL-3 storage posttreatment. These preclinical animal studies demonstrate the dose-dependent clearance of tissue and plasma GL-3 by administered alpha-Gal A, thereby providing the in vivo rationale-and the critical pharmacokinetic and pharmacodynamic data-for the design of enzyme-replacement trials in patients with Fabry disease.     

Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

Effects of dexamethasone on K(+)-evoked glutamate release from rat hippocampal slices

Dexamethasone (DEX) at physiologically elevated (stress) concentration (1 µM) decreased K⁺-evoked glutamate release from rat hippocampal slices under superfusion in the presence of Ca²⁺. On the contrary 10 µM DEX increased this K⁺-evoked glutamate release while 0.1 µM DEX had no effect. The glucocorticoid antagonist for the classic receptor, RU 486, completely reversed the effect of 1 µM DEX. Actinomycin D had no effect. Dexamethasone at 1 µM had no effect on the Ca²⁺-independent (10 µM Mg²⁺ replacing 1 mM Ca²⁺) K⁺-evoked glutamate release. Dexamethasone at 1 µM or 10 µM had no effect on the phosphate-activated glutaminase—the key enzyme for the biosynthesis of neurotransmitter glutamate. These results suggest that the effect of DEX on K⁺-evoked glutamate release: (i) depends on its concentration; (ii) is exerted on the Ca²⁺-dependent (neurotransmitter release), at least at physiological stress concentrations; and (iii) is exerted via the classical receptor but is nongenomic.     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.