Reduced presynaptic vesicle stores mediate cellular and network plasticity defects in an early-stage mouse model of Alzheimer's disease.

Background: Identifying effective strategies to prevent memory loss in AD has eluded researchers to date, and likely reflects insufficient understanding of early pathogenic mechanisms directly affecting memory encoding. As synaptic loss best correlates with memory loss in AD, refocusing efforts to identify factors driving synaptic impairments may provide the critical insight needed to advance the field. In this study, we reveal a previously undescribed cascade of events underlying pre and postsynaptic hippocampal signaling deficits linked to cognitive decline in AD.

Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46.

Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling.

Sedlin controls the ER export of procollagen by regulating the Sar1 cycle.

Newly synthesized proteins exit the endoplasmic reticulum (ER) via coat protein complex II (COPII) vesicles. Procollagen (PC), however, forms prefibrils that are too large to fit into typical COPII vesicles; PC thus needs large transport carriers, which we term megacarriers. TANGO1 assists PC packing, but its role in promoting the growth of megacarriers is not known.

Properties of connexin 46 hemichannels in dissociated lens fiber cells.

Purpose: To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses.

Methods: Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence.

COMP mutations: domain-dependent relationship between abnormal chondrocyte trafficking and clinical PSACH and MED phenotypes.

Mutations in cartilage oligomeric matrix protein (COMP) produce clinical phenotypes ranging from the severe end of the spectrum, pseudoachondroplasia (PSACH), which is a dwarfing condition, to a mild condition, multiple epiphyseal dysplasia (MED). Patient chondrocytes have a unique morphology characterized by distended rER cisternae containing lamellar deposits of COMP and other extracellular matrix proteins. It has been difficult to determine why different mutations give rise to variable clinical phenotypes.

Role of the N-terminus in permeability of chicken connexin45.6 gap junctional channels.

Previous studies have shown that gap junctional channels formed from the lens connexins Cx50 (or its chicken orthologue, Cx45.6) and Cx43 exhibit marked differences in transjunctional voltage gating and unitary conductance. In the present study, we used the negatively charged dye, Lucifer Yellow (LY), to examine and compare quantitative differences in dye transfer between pairs of HeLa cells stably transfected with Cx45.

The CMP-sialic acid transporter is localized in the medial-trans Golgi and possesses two specific endoplasmic reticulum export motifs in its carboxyl-terminal cytoplasmic tail.

The addition of sialic acid to glycoproteins and glycolipids requires Golgi sialyltransferases to have access to their glycoconjugate substrates and nucleotide sugar donor, CMP-sialic acid. CMP-sialic acid is transported into the lumen of the Golgi complex through the CMP-sialic acid transporter, an antiporter that also functions to transport CMP into the cytosol. We localized the transporter using immunofluorescence and deconvolution microscopy to test the prediction that it is broadly distributed across the Golgi stack to serve the many sialyltransferases involved in glycoconjugate sialylation.

Nuclear aggresomes form by fusion of PML-associated aggregates.

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170* aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein.

Cell-type specific trafficking of expressed mutant COMP in a cell culture model for PSACH.

Pseudoachondroplasia (PSACH) is an autosomal dominant disease that mainly affects cartilage, resulting in skeletal dysplasias and early onset osteoarthritis. PSACH is caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. PSACH chondrocytes accumulate unique COMP-containing lamellar structures in an expanded rough endoplasmic reticulum (rER).

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization.

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work.

Aggrecan domains expected to traffic through the exocytic pathway are misdirected to the nucleus.

In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.

Characterization of cartilage oligomeric matrix protein (COMP) in human normal and pseudoachondroplasia musculoskeletal tissues.

Cartilage oligomeric matrix protein (COMP), the fifth member of the -thrombospondin gene family, is an extracellular matrix calcium-binding protein. The importance of COMP is underscored by the finding that mutations in COMP cause the human dwarfing condition, pseudoachondroplasia (PSACH). Here, we report the results of human tissue distribution and cell secretion studies of human COMP.

The nanomelic mutation in the aggrecan gene is expressed in chick chondrocytes and neurons.

We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK-1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS-2 domain of the cartilage-specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L-reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK-1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene.

The ins and outs of aggrecan.

Aggrecan is a large and highly complex macromolecule, uniquely structured to fill space in the extracellular matrix (ECM) of cartilage. Lethal chondrodystrophies resulting from mutations in the structural gene for aggrecan demonstrate the serious consequences of the absence of aggrecan. Other chondrodystrophies are testimony to the importance of post-translational modifications.

The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene.

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out.

The chondrodystrophy, nanomelia: biosynthesis and processing of the defective aggrecan precursor.

The lethal chicken mutation nanomelia leads to severe skeletal defects because of a deficiency of aggrecan, which is the largest aggregating chondroitin sulphate proteoglycan of cartilage. In previous work, we have demonstrated that nanomelic chondrocytes produce a truncated aggrecan precursor that fails to be secreted, and is apparently arrested in the endoplasmic reticulum (ER). In this study, we investigated the biosynthesis and extent of processing of the abnormal aggrecan precursor.

cDNA cloning of chick cartilage chondroitin sulfate (aggrecan) core protein and identification of a stop codon in the aggrecan gene associated with the chondrodystrophy, nanomelia.

We previously reported the cloning and sequencing of a 1.5-kilobase cDNA which encoded a portion of the chondroitin sulfate domain from the chick cartilage proteoglycan core protein and the localization of a species-specific monoclonal antibody epitope. Using polymerase chain reaction amplification and primer extension, cDNA clones which code for the entire proteoglycan core protein have now been obtained from a 10-day chick embryo cDNA library.

Xylosylation is an endoplasmic reticulum to Golgi event.

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al.

Topography of glycosylation and UDP-xylose production.

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred.

Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor.

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constitutive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities.

Stable heparin-producing cell lines derived from the Furth murine mastocytoma.

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels.

Subcompartments of the endoplasmic reticulum.

The endoplasmic reticulum (ER) is the largest continuous endomembrane structure in the cytoplasm. It may be viewed as a series of unique subcompartments. In this review, we examine the rough ER, nuclear envelope and several smooth ER subcompartments.