Amy Y. Huang

Niemann-Pick type C (NPC) disease is predominantly caused by mutations in the NPC1 protein that affect intracellular cholesterol trafficking and cause accumulation of unesterified cholesterol and other lipids in lysosomal storage organelles. We report the use of a series of small molecule histone deacetylase (HDAC) inhibitors in tissue culture models of NPC human fibroblasts. Some HDAC inhibitors lead to a dramatic correction in the NPC phenotype in cells with either one or two copies of the NPC1(I1061T) mutation, and for several of the inhibitors, correction is associated with increased expression of NPC1 protein. Increased NPC1(I1061T) protein levels may partially account for the correction of the phenotype, because this mutant can promote cholesterol efflux if it is delivered to late endosomes and lysosomes. The HDAC inhibitor treatment is ineffective in an NPC2 mutant human fibroblast line. Analysis of the isoform selectivity of the compounds used implicates HDAC1 and/or HDAC2 as likely targets for the observed correction, although other HDACs may also play a role. LBH589 (panobinostat) is an orally available HDAC inhibitor that crosses the blood-brain barrier and is currently in phase III clinical trials for several types of cancer. It restores cholesterol homeostasis in cultured NPC1 mutant fibroblasts to almost normal levels within 72 h when used at 40 nM. The findings that HDAC inhibitors can correct cholesterol storage defects in human NPC1 mutant cells provide the potential basis for treatment options for NPC disease.

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane and became enriched in a subapical/apical recycling compartment. Most Alexa 488-HDL was rapidly recycled to the basolateral cell surface and released from cells. Within 30 min of chase at 37 degrees C, approximately 3% of the initial cell-associated Alexa 488-HDL accumulated in the biliary canaliculus (BC) formed at the apical pole of polarized HepG2 cells. Even less Alexa 488-HDL was transported to late endosomes or lysosomes. The fluorescent cholesterol analog dehydroergosterol (DHE) incorporated into Alexa 488-HDL was delivered to the BC within a few minutes, independent of the labeled apolipoprotein. This transport did not require metabolic energy and could be blocked by antibodies against SR-BI. The fraction of cell-associated DHE transported to the BC was comparable when cells were incubated with either Alexa 488-HDL containing DHE or with DHE bound to methyl-beta-cyclodextrin. We conclude that rapid, nonvesicular transport of sterol to the BC and efficient recycling of HDL particles underlies the selective sorting of sterol from HDLs in hepatocytes.

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as anin vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or β-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591–2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types. Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as anin vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or β-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591–2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types. carbamylcholine brefeldin A proline-rich protein parotid secretory protein trans-Golgi network SNAP receptor where SNAP is soluble NSF attachment protein polyacrylamide gel electrophoresis Salivary acinar cells are highly polarized epithelial cells that are specialized for the secretion of the macromolecular, electrolyte, and fluid components of saliva. Macromolecular secretion is achieved by the intracellular transport pathway, culminating in exocytosis of post-Golgi carriers at the apical plasma membrane. In contrast, export of electrolytes and fluid is achieved by a transepithelial flux of ions and passive flow of water mediated by specific membrane-associated pumps and channels. Secretion is mostly neurally regulated with the discharge of macromolecules and of electrolytes and fluid being controlled differentially (1Castle D. Castle A. Crit. Rev. Oral. Biol. Med. 1998; 9: 4-22Crossref PubMed Scopus (99) Google Scholar). In the parotid gland, the rate of macromolecular (mostly protein) secretion is mainly controlled by sympathetic stimulation through β-adrenergic receptors. Rates of fluid and electrolyte secretion are mainly controlled by parasympathetic stimulation through muscarinic acetylcholine receptors and, to a lesser extent, sympathetic stimulation through α-adrenergic receptors (see Refs. 2Asking B. Acta Physiol. Scand. 1985; 124: 535-542Crossref PubMed Scopus (33) Google Scholar, 3Asking B. Gjorstrup P. Acta Physiol. Scand. 1980; 109: 407-413Crossref PubMed Scopus (17) Google Scholar, 4Asking B. Gjorstrup P. Acta Physiol. Scand. 1980; 109: 415-420Crossref PubMed Scopus (4) Google Scholar and reviewed in Refs. 5Baum B.J. J. Dent. Res. 1987; 66: 628-632Crossref PubMed Google Scholar and 6Emmelin N. J. Dent. Res. 1987; 66: 509-517Crossref PubMed Scopus (119) Google Scholar). 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Biol. 1998; PubMed Scopus Google Scholar). and in the signaling pathways acinar cells to the and composition of a The secretory of acinar cells to two that are by the and of periods of or secretion in the and of the during discharge of and of Resting secretion is at in by low parasympathetic stimulation N. J. Dent. Res. 1987; 66: 509-517Crossref PubMed Scopus (119) Google Scholar). been in the pathways and of protein secretion by acinar cells of the parotid gland as they to the of resting and By using in vitro of parotid in a with we to pathways that export the salivary pathways and two are (1Castle D. Castle A. Crit. Rev. Oral. Biol. Med. 1998; 9: 4-22Crossref PubMed Scopus (99) Google Scholar). The pathways are not by secretory and and to be they for of the secretory protein a The pathway is the constitutive-like pathway, which has been observed in and cells and has been from the secretory after through the and has been deduced to originate by vesicular budding that is to the of secretory P. Castle D. J. 1998; PubMed Scopus Google Scholar). The secretory composition of the constitutive-like pathway is in proteins that are by the of M. Castle J. Cell Biol. 1987; PubMed Scopus Google Scholar, M. Castle Castle J. Biol. 1989; PubMed Google Scholar). A pathway at the of constitutive-like proteins a composition the in secretory and it may granule exocytosis. the of of which has been in M. Castle J. Cell Biol. 1987; PubMed Scopus Google Scholar, M. Castle Castle J. Biol. 1989; PubMed Google Scholar, Castle J. Cell Sci. 109: Google is of significant with to to resting the two stimulated the is secretory granule exocytosis, which we to as the regulated pathway and which for secretory protein The other is the minor regulated pathway, which for of a of secretory is from granule exocytosis by secretory proteins that been in and by secretory which that of the constitutive-like the minor regulated pathway is to stimulation is granule exocytosis and is by both cholinergic and β-adrenergic Castle J. Cell Sci. 109: Google Scholar). resting secretion in situ is by low level parasympathetic we in the minor regulated pathway serves as that is the and we show that granule exocytosis to resting secretion in from where intracellular of salivary proteins is In the minor regulated pathway in acinar we for common with the constitutive-like pathway by vesicular budding from immature and for from the constitutive-like pathway at a brefeldin A-sensitive branch point. trafficking of the minor regulated pathway to the cell to be whereas the constitutive-like pathway to be route through a distal to be rats from in the and after ad libitum or from as for of parotid lobules was from from A was from and wortmannin was from in and at The of the specific of wortmannin was by to in cells J. C. D. M. J. J. Biol. PubMed Scopus Google Scholar). and proline-rich protein in the been Castle J. Biol. PubMed Google Scholar, Castle J. Cell Biol. PubMed Scopus Google Scholar). A parotid secretory protein was by procathepsin which both and of the was from from was in by and lobules from parotid glands in with for and and in as Castle J. Cell Sci. 109: Google Scholar). Although at of at for to proteins in the trans-Golgi network P. J. Biol. PubMed Scopus Google to to for and (see in was and with or of transport as the of the in was with and and for of P. Scopus Google and for and proteins with Castle J. Cell Sci. 109: Google Scholar, Castle J. Cell Biol. PubMed Scopus Google Scholar). of intracellular and secretion of and was to a and for or as of and of of and of with of protein in a at with protein in and for and as of parotid lobules was as Castle J. Cell Biol. PubMed Scopus Google Scholar). by the J. PubMed Scopus Google in at and The in and of primary and in and and by of and stimulated for by and in in with in and in to with and for and with and with and parotid and cannulated the of the in by in at of two in the The collected from the and they for and by and In the secretory pathways that are in parotid acinar we of the secretory In we the intracellular transport and discharge of the acinar protein in and two other proline-rich protein and parotid secretory protein be in the in proteins are in the acinar cell Furthermore, of the proteins in the acinar the or in A. M. Castle, B. and J. D. Castle, these proteins secretory pathways are the of intracellular in acinar cells and not for secretory it was in parotid acinar cells that secretory to be discharged in of with being S. M. J. Cell Biol. PubMed Scopus Google Scholar). we and of secretory to the constitutive-like and minor regulated pathways in parotid and to from granule exocytosis as that export proteins M. Castle J. 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Gjorstrup P. Acta Physiol. Scand. 1980; 109: 407-413Crossref PubMed Scopus (17) Google Scholar). be in the composition of proteins low doses of carbachol that vitro with and that resting secretion is from the constitutive-like and minor regulated pathways and that the pathways are are and the in parotid by as a of secretion Arch. Biol. PubMed Scopus Google Scholar). 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