Distended Endoplasmic Reticulum Research Articles

Novel compound heterozygous variants of the SEC23A gene in a Chinese family with cranio-lenticulo-sutural dysplasia based on data from a large cohort of congenital cataract patients

BackgroundCranio-lenticulo-sutural dysplasia (CLSD) is a rare dysmorphic syndrome characterized by skeletal dysmorphism, late-closing fontanels, and cataracts. CLSD is caused by mutations in the SEC23A gene (OMIM# 607812) and can be inherited in either an autosomal dominant or autosomal recessive pattern. To date, only four mutations have been reported to cause CLSD. This study aims to identify the disease-causing variants in a large cohort of congenital cataract patients, to expand the genotypic and phenotypic spectrum of CLSD, and to confirm the association between SEC23A and autosomal recessive CLSD (ARCLSD).MethodsWe collected detailed medical records and performed comprehensive ocular examinations and whole-exome sequencing (WES) on 115 patients with congenital cataracts. After suspecting that a patient may have CLSD based on the sequencing results, we proceeded to conduct transmission electron microscopy (TEM) on the cultured skin fibroblasts. The clinical validity of the reported gene-disease relationships for the gene and the disease was evaluated using the ClinGen gene curation framework.ResultsTwo novel compound heterozygous variants (c.710A > C p.Asp237Ala, c.1946T > C p.Leu649Pro) of the SEC23A gene, classified as variant of uncertain significance, were identified in the proband with skeletal, cardiac, ocular, and hearing defects. The observation of typical distended endoplasmic reticulum cisternae further supported the diagnosis of CLSD. Application of the ClinGen gene curation framework confirmed the association between SEC23A and ARCLSD.ConclusionThis study expands the genotypic and phenotypic spectrum of CLSD, proposes TEM as a supplemental diagnostic method, and indicates that congenital cataracts are a typical sign of ARCLSD.

Endoplasmic Reticulum Stress Regulates Scleral Remodeling in a Guinea Pig Model of Form-Deprivation Myopia.

Purpose This study aimed to investigate the role of endoplasmic reticulum (ER) stress in scleral remodeling in a guinea pig model of form-deprivation myopia (FDM). Methods Guinea pigs were form deprived to induce myopia. ER ultrastructural changes in the sclera were examined by transmission electron microscopy (TEM). The protein levels of ER stress chaperones, including GRP78, CHOP, and calreticulin (CRT), were analyzed by western blotting at 24 hours, 1 week, and 4 weeks of FD. Scleral fibroblasts from guinea pigs were cultured and exposed to the ER stress inducer tunicamycin (TM) or the ER stress inhibitor 4-phenylbutyric acid (4-PBA). CRT was knocked down by lentivirus-mediated CRT shRNA transfection. The expression levels of GRP78, CHOP, TGF-β1, and COL1A1 were analyzed by qRT-PCR or western blotting. Results The sclera of FDM eyes exhibited swollen and distended ER at 4 weeks, as well as significantly increased protein expression of GRP78 and CRT at 1 week and 4 weeks, compared to the sclera of the control eyes. In vitro, TM induced ER stress in scleral fibroblasts, which was suppressed by 4-PBA. The mRNA expression of TGF-β1 and COL1A1 was upregulated after TM stimulation for 24 hours, but downregulated for 48 hours. Additionally, change of TGF-β1 and COL1A1 transcription induced by TM was suppressed by CRT knockdown. Conclusions ER stress was an important modulator which could influence the expression of the scleral collagen. CRT might be a new target for the intervention of the FDM scleral remodeling process.

Blocking the Bromodomains Function Contributes to Disturbances in Alga Chara vulgaris Spermatids Differentiation.

Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 μM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.

Upregulation of endoplasmic reticulum stress is associated with diaphragm contractile dysfunction in a rat model of sepsis.

Sepsis often causes diaphragm contractile dysfunction. Endoplasmic reticulum (ER) stress has been implicated in muscle contractile dysfunction. However, it remains unknown if ER stress occurs in the diaphragm during sepsis. In the present study, rats were divided into 4groups and received placebo or one of three durations of endotoxin treatment (24, 48h and 7days). Isometric contractile force of the diaphragm was measured and lung wet-to-dry ratio (W/D) was calculated. Hematoxylin and eosin (H&E) staining of lung tissue was performed and electron microscopy assessed ER damage in the diaphragm during sepsis. The mRNA and protein expression of glucose‑regulated protein 78kDa (GRP78), glucose-regulated protein 94kDa (GRP94), C/EBP homologous protein (CHOP), endoplasmic reticulum protein 44 (ERP44), protein disulfide-isomerase like protein (ERP57) and protein disulfide isomerase family A member 4 (ERP72) in diaphragm muscles were measured using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The level of cleaved caspase-12 was analyzed by western blot analysis. The results demonstrated that sepsis increased lung W/D. H&E staining revealed that sepsis caused alveolar congestion, hemorrhage and rupture. Swollen and distended ER was observed using electron microscopy during sepsis and decreased diaphragm contractile function was also observed. The expression levels of ER stress markers (GRP78, GRP94, CHOP, ERP44, ERP57 and ERP72) and the level of cleaved caspase‑12 were significantly elevated in septic rats compared with control rats, particularly in the 48h group. In conclusion, the present study indicated that weakened diaphragm contraction and damaged ER in septic rats was associated with increased expression of ER stress markers.

Accumulation of Laminin Monomers inDrosophilaGlia Leads to Glial Endoplasmic Reticulum Stress and Disrupted Larval Locomotion

The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, β, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. We demonstrate that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the β subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. We found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits. This research presents a new perspective on how mutations in the extracellular matrix protein laminin cause severe consequences in glial wrapping and function. Glial-specific loss of the β or γ laminin subunit disrupted glia morphology and led to ER expansion and stress due to retention of other subunits. The retention of the unpaired laminin subunit was key to the glial disruption as loss of Tango1 blocked secretion of the complete laminin trimer but did not lead to glial or locomotion defects. The effects were observed in the perineurial glia that envelope the peripheral and central nervous systems, providing evidence for the importance of this class of glia in supporting nervous system function.

Modeling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cells.

Type II collagen is a major component of cartilage. Heterozygous mutations in the type II collagen gene (COL2A1) result in a group of skeletal dysplasias known as Type II collagenopathy (COL2pathy). The understanding of COL2pathy is limited by difficulties in obtaining live chondrocytes. In the present study, we converted COL2pathy patients' fibroblasts directly into induced chondrogenic (iChon) cells. The COL2pathy-iChon cells showed suppressed expression of COL2A1 and significant apoptosis. A distended endoplasmic reticulum (ER) was detected, thus suggesting the adaptation of gene expression and cell death caused by excess ER stress. Chondrogenic supplementation adversely affected the chondrogenesis due to forced elevation of COL2A1 expression, suggesting that the application of chondrogenic drugs would worsen the disease condition. The application of a chemical chaperone increased the secretion of type II collagen, and partially rescued COL2pathy-iChon cells from apoptosis, suggesting that molecular chaperons serve as therapeutic drug candidates. We next generated induced pluripotent stem cells from COL2pathy fibroblasts. Chondrogenically differentiated COL2pathy-iPS cells showed apoptosis and increased expression of ER stress-markers. Finally, we generated teratomas by transplanting COL2pathy iPS cells into immunodeficient mice. The cartilage in the teratomas showed accumulation of type II collagen within cells, a distended ER, and sparse matrix, recapitulating the patient's cartilage. These COL2pathy models will be useful for pathophysiological studies and drug screening.

Toxic responses in rat embryonic cells to silver nanoparticles and released silver ions as analyzed via gene expression profiles and transmission electron microscopy

After exposing rat embryonic cells to 20 μg/mL of silver nanoparticle (NP) suspension and their released ions for different time periods, silver nanoparticles were found in cellular nuclei, mitochondria, cytoplasm and lysosomes by transmission electron microscopy (TEM). We also observed mitochondrial destruction, distension of endoplasmic reticulum and apoptotic bodies. Global gene expression analysis showed a total of 279 genes that were up-regulated and 389 genes that were down-regulated in the silver-NP suspension exposure group, while 3 genes were up-regulated and 41 genes were down-regulated in the silver ion exposure group. Further, the GO pathway analysis suggested that these differentially expressed genes are involved in several biological processes, such as energy metabolism, oxygen transport, enzyme activities, molecular binding, etc. It is possible that inhibition of oxygen transport is mediated by the significant down-regulation of genes of the globin family, which might play an important role in silver ion-induced toxicity. KEGG pathway analysis showed that there were 23 signal pathways that were affected in the cells after exposure to silver-NP suspension, but not silver ion alone. The most significant change concerned inflammatory signal pathways, which were only found in silver-NP suspension exposed cells, indicating that inflammatory response might play an important role in the mechanism(s) of silver-NP-induced toxicity. The significant up-regulation of matrix metalloproteinases 3 and 9 suggests that silver NPs could induce extracellular matrix degradation via an inflammatory signaling pathway. The significant up-regulation of secretory leukocyte peptidase inhibitor and serine protease inhibitor 2c was considered to be an embryonic cellular defense mechanism in response to silver-NP-induced inflammation.

ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats.

To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis. Forty rats were randomly divided into two groups: a control group and a diabetic group. Diabetes was induced by intraperitoneal injection of 60 mg/kg of streptozotocin. Gastric emptying was determined at the 4(th) and 12(th) week. The ultrastructural changes in gastric smooth muscle cells (SMCs) were investigated by transmission electron microscopy. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis of SMCs. Expression of the ER stress marker, glucose-regulated protein 78 (GRP78), and the ER-specific apoptosis mediator, caspase-12 protein, was determined by immunohistochemistry. Gastric emptying was significantly lower in the diabetic rats than in the control rats at the 12(th) wk (40.71% ± 2.50%, control rats vs 54.65% ± 5.22%, diabetic rats; P < 0.05). Swollen and distended ER with an irregular shape was observed in gastric SMCs in diabetic rats. Apoptosis of gastric SMCs increased in the diabetic rats in addition to increased expression of GRP78 and caspase-12 proteins. ER stress and ER stress-mediated apoptosis are activated in gastric SMCs in diabetic rats with gastroparesis.

Development of cycad ovules and seeds. 2. Histological and ultrastructural aspects of ontogeny of the embryo in Encephalartos natalensis (Zamiaceae)

Development of the embryo of Encephalartos natalensis from a rudimentary meristematic structure approximately 700 μm in length extends over 6 months after the seed is shed from the strobilus. Throughout its development, the embryo remains attached to a long suspensor. Differentiation of the shoot meristem flanked by two cotyledonary protuberances occurs over the first 2 months, during which peripheral tannin channels become apparent. Tannins, apparently elaborated by the endoplasmic reticulum, first accumulate in the large central vacuole and ultimately fill the channel. By the fourth month of development, the root meristem is apparent and procambial tissue forming discrete vascular bundles can be discerned in the elongating cotyledons. Between 4 and 6 months, mucilage ducts differentiate; after 6 months, when the seed becomes germinable, the embryo is characterised by cotyledons far longer than the axis. Shoot and root meristem cells remain ultrastructurally similar throughout embryo ontogeny, containing small vacuoles, many well-differentiated mitochondria and endoplasmic reticulum (ER) profiles, abundant polysomes, plastids containing small starch deposits and Golgi bodies. Unusually, however, Golgi bodies are infrequent in other cells including those elaborating mucilage which is accumulated in distended ER and apparently secreted into the duct lumen directly by ER-derived vesicles. The non-meristematic cells accumulate massive starch deposits to the exclusion of any protein bodies and only very sparse lipid, features which are considered in terms of the prolonged period of embryo development and the high atmospheric oxygen content of the Carboniferous Period, when cycads are suggested to have originated.

Ultrastructural changes in photorejuvenation induced by photodynamic therapy in a photoaged mouse model

Photoaging is defined as premature aging of the skin induced by ultraviolet (UV) irradiation. Photodynamic therapy (PDT) has been shown to be a non-invasive but effective technique for photoaged skin. We observed histological and ultrastructural changes of photoaging and photorejuvenating effects of 5-aminolevulinic acid (ALA)-PDT in a UV-irradiated mice model. A total of 20 mice were divided into a control (group A) group and a UV-irradiated (photoaging) group. The photoaging group was divided according to the following interventions: photoaging only (group B), ALA application only (group C), light-emitting diode (LED) irradiation only (group D), and ALA-PDT with a light dose of 20 J/cm(2) (group E). Serial skin biopsies were performed from day 2 to day 21, and transmission electron microscopic (TEM) studies were performed. After UV irradiation, the amount of dermal collagen fibers decreased, and the quantity of elastotic materials increased. Following ALA-PDT application, the amount of collagen fibers increased from day 2 to day 21 and the increased elastotic materials during the photoaging period were normalized. With TEM, the decreased collagen fibers during photoaging were restored after PDT application. Also, distended dermal fibroblasts with distended endoplasmic reticulum by UV irradiation were normalized after PDT application. This study provides histologic evidence of the beneficial effects of ALA-PDT, even in photodamaged skin. ALA-PDT induces deposition of collagen in the dermis, normalizes elastotic materials which were induced by photoaging and may even have a direct effect on the normalization of the morphology of fibroblasts.

Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling

Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.

Association with Endoplasmic Reticulum Promotes Proteasomal Degradation of GADD34 Protein

Stress-induced endogenous and ectopically expressed GADD34 proteins were present both in the cytoplasm and in membranes, with their membrane association showing similar biochemical properties. Deletion of N-terminal sequences in GADD34-GFP proteins highlighted an amphipathic helix, whose hydrophobic surface, specifically valine 25 and leucine 29, mediated endoplasmic reticulum (ER) localization. Substitution of leucines for three arginines on the polar surface indicated that the same helix also mediated the association of GADD34 with mitochondria. Fluorescence protease protection and chemical modification of cysteines substituted in the membrane-binding domain pointed to a monotopic insertion of GADD34 into the outer layer of the ER membrane. Fluorescence recovery after photobleaching showed that ER association retards the mobility of GADD34 in living cells. Both WT GADD34 and the mutant, V25R, effectively scaffolded the α-isoform of protein phosphatase-1 (PP1α) and enabled eIF2α dephosphorylation. However, the largely cytosolic V25R protein displayed a reduced rate of proteasomal degradation, and unlike WT GADD34, whose ectopic expression resulted in a dilated or distended ER, V25R did not modify ER morphology. These studies suggested that the association of with ER modulates intracellular trafficking and proteasomal degradation of GADD34, and in turn, its ability to modify ER morphology.

Role of autophagy in β‐cell function and mass

Type 2 diabetes (T2D) is characterized by decreased insulin secretion and action. Decreased insulin secretion results from a reduction in pancreatic β-cell mass and/or function. Apoptosis, oxidative stress, mitochondrial dysfunction and endoplasmic reticulum (ER) stress responses including JNK activation have been suggested as mechanisms for the changes of pancreatic β-cells in T2D; however, the underlying causes were not clearly elucidated. Autophagy is an intracellular process that plays crucial roles in cellular homeostasis through degradation and recycling of organelles. We have reported increased apoptosis and decreased proliferation of β-cells with resultant reduction in the β-cell mass in β-cell-specific autophagy-deficient mice. Morphological analysis of β-cells revealed accumulation of ubiquitinated proteins, swollen mitochondria and distended ER. Insulin secretory function ex vivo was also impaired. As a result, β-cell-specific autophagy-deficient mice showed hypoinsulinaemia and hyperglycaemia. These results suggested that autophagy is necessary to maintain the structure, mass and function of pancreatic β-cells. In addition, as autophagy may play a protective role against ER stress and rejuvenates organelle function, impaired autophagy may lead to mitochondrial dysfunction and ER stress, which have been implicated as potential causes of insulin resistance. Therefore, in addition to β-cell homeostasis, dysregulated autophagy may possibly be involved in diverse aspects of the pathogenesis of diabetes.

PERK (EIF2AK3) Regulates Proinsulin Trafficking and Quality Control in the Secretory Pathway

OBJECTIVELoss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of β-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient β-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes.RESEARCH DESIGN AND METHODSER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2+/Akita mutant mice were used as a model system to test the role of PERK in ERAD.RESULTSWe report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes.CONCLUSIONSPERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting β-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.

Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

H Syndrome: Recently Defined Genodermatosis With Distinct Histologic Features. A Morphological, Histochemical, Immunohistochemical, and Ultrastructural Study of 10 Cases

This study analyzes the histopathological findings in H syndrome, a recently recognized autosomal recessive genodermatosis characterized by indurated, hyperpigmented, and hypertrichotic skin in well-defined anatomical areas accompanied by various systemic manifestations. So far, descriptions of the histopathological skin changes in this disorder, as reported in a few small case series, were inconsistent, leading to diverse clinical interpretations. In an attempt to define standardized, diagnostic, morphological criteria that will distinguish this disorder from other fibrosing conditions, we studied skin biopsies from 10 patients with H syndrome. The characteristic morphology included widespread fibrosis (moderate in dermis and severe in subcutis); striking mononuclear infiltrates consisting mainly of monocyte-derived cells (small CD68 histiocytes and CD34 and FXIIIa dendrocytes) and plasma cells; and thickened, fragmented, and partially calcified elastic fibers, admixed with well-formed psammoma bodies, a previously unrecognized feature in nonneoplastic skin and subcutaneous conditions. In addition, the ultrastructure of CD68 small histiocytes exhibited distended endoplasmic reticulum and scarcity of lysosomes, features typical for fibroblasts but unusual for histiocytes. These unusual findings in the histiocytes pose a question as to their possible role in the fibrotic cascade in this disorder. We conclude that the above findings are essential for the diagnosis of H syndrome and that incisional biopsies are mandatory for recognition of the full spectrum of histopathological findings.

Autophagy in Diabetes

Diabetes mellitus is characterized by decreased insulin secreti on and action. Decreased insulin secretion results from a reduction in mass and/or function of pancreatic β-cells. Apoptosis, oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress responses have been suggested as mechanisms for the changes in β -cells in type 2 diabetes; however, the underlying causes have not been clearly elucidated. Autophagy is an intracellular process that maintains cellular homeostasis through degradation and recycling of organelles. Recently, we reported reduction of β-cell mass in autophagy-deficient mice. Pancreatic insulin content was also decreased due to the decreased β-cell mass and the reduced number of insulin granules. Morphological analysis of these β-cells revealed an accumulation of ubiquitinated proteins, swollen mitochondria, and distended ER. Insulin secretory function ex vivo was also impaired. As a result, autophagy-deficient mice showed hypoinsulinemia and hyperglycemia. These results suggested that autophagy is necessary to maintain the structure, mass and function of β-cells. In addition, as autophagy may play a protective role against ER stress and rejuvenate organelle function, impaired autophagy may lead to mitochondrial dysfunction and ER stress, which have been implicated as causes of insulin resistance. Therefore, in addition to β-cell homeostasis, dysregulated autophagy may possibly be involved in insulin resistance. (Korean Diabetes J 33:453-457, 2009)

Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development

The COPII coat assembles on endoplasmic reticulum membranes to coordinate the collection of secretory cargo with the formation of transport vesicles. During COPII assembly, Sar1 deforms the membrane and recruits the Sec23-Sec24 complex (Sec23/24), which is the primary cargo-binding adaptor for the system, and Sec13-Sec31 (Sec13/31), which provides a structural outer layer for vesicle formation. Here we show that Sec13 depletion results in concomitant loss of Sec31 and juxtanuclear clustering of pre-budding complexes containing Sec23/24 and cargo. Electron microscopy reveals the presence of curved coated profiles on distended endoplasmic reticulum, indicating that Sec13/31 is not required for the generation or maintenance of the curvature. Surprisingly, export of tsO45-G-YFP, a marker of secretory cargo, is unaffected by Sec13/31 depletion; by contrast, secretion of collagen from primary fibroblasts is strongly inhibited. Suppression of Sec13 expression in zebrafish causes defects in proteoglycan deposition and skeletal abnormalities that are grossly similar to the craniofacial abnormalities of crusher mutant zebrafish and patients with cranio-lenticulo-sutural dysplasia. We conclude that efficient coupling of the inner (Sec23/24) and outer (Sec13/31) layers of the COPII coat is required to drive the export of collagen from the endoplasmic reticulum, and that highly efficient COPII assembly is essential for normal craniofacial development during embryogenesis.

N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite of animal cells. Infection of humans is common and may result in devastating disease, especially in immunocompromised individuals. Despite previous reports that N-glycosylation of proteins may be a rare post-translational modification in this and related organisms, we demonstrate that it is actually quite prevalent in Toxoplasma. N-Glycosylation is completely inhibited by treatment of parasites with tunicamycin, but this does not appear to exert its major effect on the parasites until they have egressed from their host cells. Although the tunicamycin-treated parasites appear structurally normal at this time they are not motile and mostly incapable of invading new host cells. The few tunicamycin-treated parasites that do invade are severely affected in their ability to replicate and accumulate with a distended endoplasmic reticulum, deformed nuclei, and without recognizable late secretory organelles. We provide experimental evidence that indicate that Toxoplasma N-glycans differ structurally from those in other eukaryotes.

Effects of CDB-4022 on Leydig Cell Function in Adult Male Rats1

CDB-4022, an indenopryridine, suppresses spermatogenesis and decreases inhibin secretion in adult male rats. In the present study, we investigated the effects of CDB-4022 on Leydig cell function. A single oral dose of CDB-4022 (2.5 mg/kg) resulted in a 2-fold decrease in serum testosterone levels after 7 days that was paralleled by a decrease in Cyp17a1 mRNA and protein levels and 17alpha hydroxylase enzymatic activity compared with vehicle-treated rats. Consistent with the lower serum testosterone levels, pituitary Lhb and Fshb mRNA levels were increased 3.2- and 2.3-fold, respectively, by CDB-4022 treatment. Ultrastructural analysis of pituitary gonadotrophs showed distended endoplasmic reticulum (ER) and fewer secretory granules in CDB-4022-treated rats, characteristic of enhanced secretory activity. Conversely, CDB-4022 increased serum progesterone levels, testicular Star mRNA and protein expression, and the number of Leydig cells per testis. Serum inhibin B levels were undetectable in CDB-4022-treated rats, while serum activin A levels were similar to controls, indicating that the CDB-4022-treated rats have an elevated activin A:inhibin B ratio. In the presence of hCG stimulation, activin A directly suppressed testosterone secretion but enhanced progesterone secretion from rat Leydig cell primary cultures. Likewise, treatment of MA-10 cells with activin A was found to enhance cAMP-stimulated progesterone secretion and STAR expression. Together, our data indicate that CDB-4022 treatment inhibits CYP17A1 and stimulates STAR expression, thereby decreasing testosterone but increasing progesterone production. We propose that unopposed actions of activin A most likely contribute to the steroid profile in rats after CDB-4022 treatment. Our findings establish CDB-4022 as a new model to examine intratesticular control mechanisms that modulate Leydig cell gene expression and function.