Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
Niamh X. Cawley, PhD, Staff Scientist
Marjorie Gondre-Lewis, PhD, Research Fellow
Irina Arnaoutova, PhD, Postdoctoral Fellow
Taeyoon Kim, PhD, Postdoctoral Fellow
Hisatsugu Koshimizu, PhD, Postdoctoral Fellow
Josh Park, PhD, Postdoctoral Fellow
Andre Phillips, PhD, Postdoctoral Fellow
Alicja Woronowizc, MD, PhD, Postdoctoral Fellow
Tulin Yanik, PhD, Postdoctoral Fellow
Hong Lou, MD, Senior Research Assistant
Annahita Sarcon, BS, Postbaccalaureate Fellow
Meera Sridhar, BS, Postbaccalaureate Fellow

We study the cell biology of endocrine and neuroendocrine cells. In particular, we direct our efforts to (1) investigating the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes; (2) uncovering mechanisms involved in the regulation of dense-core secretory granule biogenesis; and (3) determining the physiological roles of the prohormone processing enzyme carboxypeptidase E. Our work has led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway and the identification of players and mechanisms that control secretory granule biogenesis and transport in endocrine and neuroendocrine cells and neurons. Such studies in cell lines, primary cell cultures, and mouse models have provided a better understanding of diabetes, obesity, and cholesterol deficiency and of diseases related to defects in hormone and neuropeptide targeting and synaptic transmission.

Mechanism of sorting pro-neuropeptides, neurotrophins, and their processing enzymes to the regulated secretory pathway

Cawley, Lou, Arnaoutova, Sridhar, Loh; in collaboration with Baum, Chow

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for the processing, storage, and release of active proteins and peptides in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin, and brain-derived neurotrophic factor (BDNF) to the RSP. We showed that these pro-proteins undergo homotypic oligomerization as a concentration step as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN), where they are sorted into the dense-core granules of the RSP for processing by prohormone convertases (PCs) and carboxypeptidase E (CPE) and for secretion. Site-directed mutagenesis identified a consensus sorting motif consisting of two exposed acidic residues located 12-15Å apart and two hydrophobic residues located 5-7Å away from the acidic residues, which are all necessary for sorting to the RSP. We found such a motif in BDNF, which is secreted in an activity-dependent manner, but not in constitutively secreted nerve growth factor (NGF), which lacks one amino acid residue of the motif. Mutagenic introduction of the missing residue (Val20Glu) redirected NGF to the RSP, underscoring the importance of the sorting motif in targeting to the RSP.

We identified the transmembrane form of CPE as an RSP sorting receptor that was specific for the sorting signal of POMC, proinsulin, and BDNF. Two acidic residues in the prohormone/pro-BDNF sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor CPE to effect sorting to the RSP. Transfection of a mutant CPE with R255 and K260 mutated to Ala caused missorting of co-transfected POMC to the constitutive pathway, indicating that sorting to the RSP occurs when the basic residues in the CPE sorting domain interact with the acidic residues in the POMC sorting signal. Transfection of a dominant negative CPE mutant or depletion of CPE by using siRNA caused partial missorting of proinsulin to the constitutive pathway, indicating an interaction of proinsulin and CPE to effect sorting. Furthermore, by using a CPE knockout (KO) mouse model, we showed that BDNF was not sorted to the RSP but was secreted constitutively in the animals' cortical and hippocampal neurons. Constitutive secretion of proinsulin from isolated pancreatic islets and plasma levels of proinsulin were significantly higher in CPE KO than in wild-type mice, indicating a role of CPE in intracellular trafficking of proinsulin. Furthermore, primary pituitary cultures from CPE KO mice showed poorly regulated secretion of POMC/ACTH, consistent with CPE's function as a sorting receptor. However, we also observed in a subpopulation of KO animals a similar stimulated secretion of ACTH as in wild type. This subpopulation of mice had elevated expression of secretogranin III (SgIII), a lipid raft-associated molecule found in the TGN. Work reported in the literature has shown that SgIII binds to POMC, albeit with lower affinity than to CPE, and can potentially act as a sorting receptor for POMC. Thus, at the elevated expression levels that occur in some CPE KO mice, SgIII appears to act as a compensatory sorting receptor for POMC.

The enzymes CPE, PC1, and PC2 are known to be transmembrane, secretory-vesicle proteins with an atypical membrane-spanning domain at the C-terminus and a cytoplasmic tail. We have shown that the sorting of these enzymes into granules of the RSP in neuroendocrine cells requires association of their C-terminal domain with cholesterol-glycosphingolipid-rich microdomains, which are known as lipid rafts, at the TGN. Removal of cholesterol from secretory granule membranes resulted in CPE's inability to act as a sorting receptor and bind to cargo; depleting cholesterol with lovastatin prevented sorting of CPE to the RSP. Removing the transmembrane domain of PC1/3 alone (residues 617-638) and leaving the C-terminal domain (residues 639-752) intact led to missorting of the enzyme to the constitutive pathway. Thus, translocation of the transmembrane domain of PC1/3 through cholesterol-rich lipid rafts at the TGN appears to be essential for the sorting of PC1/3, and probably PC2 and CPE as well, to the RSP.

Our knowledge of hormone sorting motifs has enabled us to engineer mutant hormones that are redirected to the constitutive pathway. We are currently expressing these hormones in salivary glands for systemic secretion, with the aim of applying such technology to gene therapeutics. Recently, we expressed a mutant form of proinsulin in murine salivary glands; the mutant form was secreted constitutively into the blood, resulting in lowered plasma glucose levels.

Absence of CART linked to obesity and low bone density

Yanik, Woronowizc, Cawley, Loh in collaboration with del Guidice, Kuhar, Marini

We investigated the sorting and processing of a mutant form of cocaine- and amphetamine-regulated transcript (CART) found in a family of obese patients. CART, found in brain, is an anorectic peptide that has several physiological effects such as inhibiting feeding and regulating energy expenditure; CART is also involved in the stress response. CART acts downstream of leptin in the obesity-controlling signaling pathway. A mutant pro-CART (Leu34Phe) was found in a 10-year-old Italian boy who has been obese since two years of age. This missense Leu34Phe mutation co-segregates in three generations of his maternal relatives along with the phenotype of severe obesity, but his father was not obese. To investigate whether these patients have mature CART, we used Protein Chip technology (Ciphergen) to analyze serum from the obese boy and affected family members from three generations as well as from an unaffected sibling and five normal controls. All members of the family bearing the (Phe34Leu) mutation showed only pro- and intermediate CART but no mature CART in their circulation. In contrast, normal humans and the unaffected sibling showed significant amounts of circulating mature CART. To determine the cellular basis for the lack of mature CART in the obese patients, we investigated the trafficking and processing of mutant CART by transfecting wild-type (WT) or mutant (Leu34Phe) CART into AtT-20 cells. While pro-CART was substantially processed to active CART, mutant pro-CART was only minimally processed to yield an intermediate form in the AtT-20 cells. Furthermore, WT CART was secreted in a regulated manner with high potassium stimulation, but mutant pro-CART/CART exhibited high basal release and no significant stimulated secretion. Immunocytochemical studies revealed that immunoreactive WT CART was primarily co-localized in punctate granules with POMC—a granule marker—in the processes of AtT-20 cells. However, about half the cells showed no punctate staining of immunoreactive mutant CART co-localized with POMC in the cell processes, indicating that mutant pro-CART was partially missorted and secreted via the constitutive pathway. The poor processing of mutant pro-CART is likely attributable to the missorting to the constitutive pathway, which lacks the appropriate processing enzymes. Thus, the missorting and lack of processing of mutant CART (Leu34Phe) provides a molecular basis for the obese phenotype in the obese patients.

We recently investigated the role of CART in bone remodeling. Using Protein Chip technology, we confirmed that, unlike WT mice, the CPE KO mice, which we had previously shown to lack plasma CART, do not have mature CART in the hypothalamus. Bone density scans revealed that both male and female CPE KO mice had lower bone density than WT mice. Furthermore, the osteoblast numbers were significantly lower and the osteoclast numbers significantly higher in the CPE KO animals. This finding is consistent with the literature showing that CART¿/¿ mice have increased osteoclast numbers, further supporting the role of CART in bone remodeling. However, the fact that osteoblast numbers in CART¿/¿ mice were not lower suggests that other neuropeptides or peptide hormones missing in the CPE KO mice may play a role in osteoblast formation or survival.

Abnormal brain morphology, behavior, and deficits in synaptic transmission in the CPE knockout mouse

Woronowizc, Cawley, Sarcon, Loh; in collaboration with Craft, Hill, Wetsel

We generated a carboxypeptidase E knockout (CPE KO) mouse by deleting exons 4 and 5 from the CPE gene and then characterized the animal's phenotype. The KO mice became obese by 10-12 weeks of age and weighed 60-80 g by 40 weeks. The null animals consumed more food overall, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than WT littermates. Fasting levels of glucose and insulin-like immunoreactivity (IR) in plasma were elevated in the KO mice at about 20 weeks; males recovered from this state by 32 weeks but females did not. At this age, the KO animals' plasma insulin-like IR, which consists primarily of pro-insulin, was 50-100 times higher than that of the WT animals. The KO mice showed impaired glucose clearance and were insulin-resistant. Plasma levels of leptin were high in the KO mice; however, the mice showed no circulating fully processed CART, a peptide that is responsive to leptin-induced feedback inhibition of feeding. In addition to their obesity and diabetes phenotypes, the KO mice were subfertile and showed deficits in GnRH processing in the hypothalamus.

Behavioral analyses revealed that the KO animals were less reactive to stimuli and exhibited lower muscle strength, coordination, and visual placing and toe-pinch reflexes. Moreover, they showed delayed learning in the water maze test. Analysis of brain sections revealed that the structure of the hippocampus in the KO mice was abnormal. Electron microscopy showed that 40 percent of the synapses examined in the hypothalamus of the mice lacked presynaptic docked synaptic vesicles. Our recent studies indicate that CPE is present in synaptic vesicles. The mice also exhibited abnormal neurotransmission from the photoreceptors to the inner retina, showing a loss of the b wave in their retinogram. We obtained similar results in mutant mice (Cpe^fat/^fat) bearing a substitution mutation in residue Ser202Pro of CPE. Immunocytochemical studies on these mice revealed that, unlike in the case of normal mice, the outer plexiform layer lacked CPE but that CPE accumulated in the outer nuclear layer, which is where the cell bodies of the photoreceptor cells are localized. Furthermore, EM studies indicated significantly fewer synaptic vesicles in the spherules (synaptic butons), suggesting a defect in transport of synaptic vesicles carrying CPE. Thus, CPE may play a role in neuronal development and migration of cells to form the hippocampus as well as in transport and movement of synaptic vesicles/peptidergic vesicles along the nerve and to the release site at specific synapses in the brain. Absence of CPE in the KO mice therefore leads to failure in neurotransmission, deficits in learning and memory, and abnormal behavior.

Role of CPE in transport of POMC and BDNF vesicles in endocrine cells and neurons

Park, Phillips, Cawley, Loh

Post-Golgi transport of hormone and BDNF vesicles for activity-dependent secretion is important in mediating endocrine function and synaptic plasticity. The previous discussion of morphological studies, which point to synaptic vesicle transport and docking abnormalities in, respectively, the retina and hypothalamus in CPE KO mice, prompted us to investigate the role of the CPE cytoplasmic tail in vesicle movement. We showed that overexpression of the CPE cytoplasmic tail diminished localization of endogenous POMC, BDNF, and fluorescence-tagged CPE in the processes of the endocrine cell line AtT-20 and hippocampal neurons. In live primary pituitary and AtT20 cell images, overexpression of the CPE tail in the cytoplasm inhibited the movement of POMC/CPE-containing vesicles to the processes. S-tagged CPE tail pulled down the endogenous microtubule-based motors dynactin (p150), dynein, and KIF1A from cytosol of AtT-20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT-20 cells. Our research has thus uncovered a novel mechanism whereby the vesicular CPE cytoplasmic tail plays a mandatory role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to microtubule-based motors for transport and activity-dependent secretion in endocrine cells and neurons. We recently demonstrated that CPE is associated not only with large dense-core vesicles but also with synaptic vesicles, suggesting that the CPE tail may also be involved in transport of synaptic vesicles.

In recent yeast two-hybrid studies, we showed that the CPE tail also interacted with γ-adducin; we then confirmed such interaction in precipitation studies in vitro. Known primarily as a component of the cytoskeleton that binds to and stabilizes F-actin and mediates actin binding to fodrin, γ-adducin has long been thought of as a structural protein. However, we recently made observations suggesting an expanded role for the protein. To assess the significance of the interaction between the CPE tail and γ-adducin in a functional system, we overexpressed the last 25 amino acids of the CPE C-terminal domain (CPE25) in the cytoplasm of AtT-20 cells. Immunocytochemical detection of γ adducin indicated a robust distribution of the protein in cell processes while a shift in γ-adducin localization largely to the cell body occurred in the presence of CPE-25. Given that overexpression of CPE-25 inhibits interaction of the cytoplasmic tail with dynactin and hence vesicle movement along the microtubules in the processes, the redistribution of γ-adducin in the presence of CPE-25 suggests that γ-adducin binds to the vesicles via the CPE cytoplasmic tail and moves with these organelles. We hypothesize that γ-adducin linked to CPE tail may participate in the tethering of ACTH vesicles to cortical actin just below the plasma membrane in readiness for priming, docking, and release upon stimulation.

Mouse models of cholesterol deficiency diseases exhibit defective secretory granule biogenesis in exocrine and endocrine cells

Gondre-Lewis, Loh; in collaboration with Parsegian, Porter

To test the importance of cholesterol in secretory granule biogenesis and packaging of granule contents in vivo, we analyzed vesicles in the pancreas of cholesterol-deficient mouse models of Smith-Lemli Opitz syndrome (SLOS) and lathosterolosis (Sc5d¿/¿). SLOS and lathosterolosis are human disorders caused by deficiencies in, respectively, 7-dehydrocholesterol reductase and lathosterol 5-desaturase, enzymes necessary for the final steps of cholesterol biosynthesis. Morphological analysis by light and electron microscopy of neonatal pancreas zymogen granules showed markedly lower numbers of granules in both SLOS and Sc5d¿/¿ than in control mice. Most of the granules present in SLOS and Sc5d¿/¿ animals were of an immature phenotype, appearing as partially formed spheres, in contrast to those in control animals. Sc5d¿/¿ exocrine pancreas lacking granules was filled with rough ER ribbons and ribosomal structures, indicating an inability to package materials into membrane-bound structures. Furthermore, in primary cultures of cholesterol-deficient secretory cells in the exocrine pancreas, granule biogenesis and regulated secretion of ¿-amylase was impaired as compared with control cells. The addition of exogenous cholesterol to these cells rescued the phenotype. Granule biogenesis was also impaired in endocrine tissues of Sc5d¿/¿ mice. We hypothesize that the defect in granule biogenesis and maturation is attributable to different physical contributions of sterols to membrane curvature. Indeed, biophysical studies indicate that 7-dehydrocholesterol and lathosterol have a lower binding rigidity than cholesterol and are therefore less able to form curvature. Thus, genetic inhibition of cholesterol synthesis in SLOS and Sc5d¿/¿ mice impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine and endocrine systems.

Regulation of secretory granule biogenesis by chromogranin A

Kim, Koshimizu, Arnaoutova, Loh

Formation of large dense-core granules (LDCGs) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered an on/off switch, chromogranin A (CgA), that controls the formation of LDCG in neuroendocrine cells. Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of LDCG and regulated secretion as well as in the degradation of granule proteins, including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild-type phenotype. In the mutant endocrine cell line 6T3, which lacks CgA, LDCGs, and regulated hormone secretion, transfection of CgA resulted in the rescue of granule biogenesis and the regulated secretory pathway. The importance of CgA in LDCG biogenesis was evident not only in cell lines but also in vivo. In an antisense mRNA transgenic mouse model deficient in CgA, we observed both quantitatively and qualitatively severe aberrant granule formation in the adrenal medulla as well as a correlation between depletion of CgA and reduction in secretory granule biogenesis. A reduction in granule proteins accompanied the reduction in secretory granule biogenesis in the adrenal medulla of the CgA-deficient transgenic animals and in 6T3 cells lacking CgA. Using 6T3 cells lacking secretory granules as a model, we showed that reduced granule protein levels were attributable to degradation at the Golgi apparatus. We therefore proposed that regulation of the stability of granule proteins at the Golgi apparatus by CgA is a focal point for control of granule biogenesis in neuroendocrine cells. In support of our hypothesis, we recently found a protease inhibitor, protease nexin-1 (PN-1), in the Golgi that is transcriptionally activated by CgA. This inhibitor is upregulated in cells actively forming LDCGs but downregulated in cells that minimally express CgA and show low levels of LDCG biogenesis. Moreover, transfection of PN-1 into 6T3 cells lacking CgA prevented LDCG protein degradation and rescued granule biogenesis. Furthermore, downregulation of expression of PN-1 by antisense RNA in 6T3 cells transfected with CgA led to enhanced degradation of granule proteins and less secretory granule formation. Recently, we showed that 6T3 cells incubated with conditioned medium from 6T3-bCgA cells or AtT-20 cells produced an increase in PN-1 mRNA and granule biogenesis. Likewise, stimulation of AtT-20 cells with high potassium resulted in an increase in PN-1 mRNA, an increase that was blocked by actinomycin D. We hypothesize that CgA, or a proteolytic fragment of CgA, may bind to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN1-mRNA transcription. We have thus uncovered a novel mechanism whereby CgA/CgA-derived peptides secreted into the medium regulate large dense-core granule biogenesis by transcriptionally activating the protease inhibitor PN-1, which stabilizes granule proteins at the TGN to increase LDCG biogenesis in endocrine cells.

Role of aquaporin 1 in hormone secretion and secretory granule biogenesis

Arnaoutova, Kim, Loh

Recently, we found that the water channel protein aquaporin 1 (AQP1), which is normally present in the plasma membrane, is also found in secretory granules of endocrine cells. Moreover, when we transfected 6T3 cells lacking CgA with CgA, which rescues granule biogenesis in 6T3 cells, we observed a significant increase in expression of AQP1 mRNA and protein. To investigate the role of AQP1 in hormone sequestration and granule biogenesis, we stably transfected AtT-20 cells with an antisense construct of AQP1 to downregulate expression of the protein. AQP1-deficient AtT-20 cells showed a dramatic reduction in secretory granule number. Pulse-chase studies demonstrated a defect in regulated secretion of the endogenous ACTH hormone as well as enhanced degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. However, the deficiency of AQP1 did not affect transcription and translation of the newly synthesized proteins in the transfected cells. AQP1 appears to be critical for the mechanism of secretory granule exocytosis. A defect in secretion leads to a feedback to downregulate secretory granule biogenesis.

COLLABORATORS

Bruce Baum, DMD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD
Nigel Birch, PhD, University of Auckland, Auckland, New Zealand
Robert Chow, PhD, University of Southern California, Los Angeles, CA
Cheryl Craft, PhD, University of Southern California, Los Angeles, CA
Emanuele del Guidice, MD, Seconda Università di Napoli, Naples, Italy
Joanna Hill, PhD, Laboratory of Behavioral Neuroscience, NIMH, Bethesda, MD
Michael Kuhar, PhD, Yerkes National Primate Center, Emory University, Atlanta, GA
Joan Marini, MD, PhD, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
Adrian Parsegian, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Forbes Porter, MD, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
William Wetsel, PhD, Duke University, Durham, NC

For further information, contact lohp@mail.nih.gov.