Mice with a targeted deletion of the nidogen-binding site of laminin gamma1 were used to study the function of the pial basement membrane in cortical histogenesis. The pial basement membrane in the mutant embryos assembled but was unstable and disintegrated at random segments. In segments with a disrupted basement membrane, radial glia cells were retracted from the pial surface, and radially migrating neurons, including Cajal-Retzius cells and cortical plate neurons, passed the meninges or terminated their migration prematurely. By correlating the disruptions in the pial basal lamina with changes in the morphology of radial glia cells, the aberrant migration of Cajal-Retzius cells, and subsequent dysplasia of cortical plate neurons, the present data establish a causal relationship of proper cortical histogenesis with the presence of an intact pial basement membrane.

The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under nondenaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons.Heparan sulfate proteoglycans (HSPGs) 1 are members of a family of cell-surface proteins with long carbohydrate chains of repeating disaccharide units (1-3). They exist as integral membrane proteins (4) or as secreted extracellular matrix proteins. HSPGs have a variety of biological functions, such as serving as molecular sieves for the ultrafiltration of blood in the kidney (5), sequestering growth factors (6 -8), lining blood vessels (9), and serving as a structural component of basal laminae. Perlecan, a large proteoglycan from Engelbreth-Holm-Swarm mouse sarcoma, was the first molecularly identified member of the extracellular HSPGs (10, 11). cDNA cloning of the murine and human perlecan showed that its protein core consists of various domains that are homologous to the low density lipoprotein receptor, laminin, NCAM, and epidermal growth factor. The perlecan core protein with over 400 kDa is one of the largest gene products known today (11,12). The second representative member of extracellular HSPGs is agrin (13, 14), a protein that was first isolated from fish electric organ for its activity to cluster acetylcholine receptors on the surface of myoblasts (15). Like perlecan, agrin is a large multi-domain protein of over 200 kDa with several laminin G, follistatin, and epidermal growth factor-like repeats (16 -18). Side by side comparison shows that the domain arrangements at t...

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/ endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level.

We have addressed the question of whether a family of axon growth-promoting molecules known as the laminins may play a role during axon regeneration in the CNS. A narrow sickle-shaped region containing a basal lamina-independent form of laminin exists in and around the cell bodies and proximal portion of the apical dendrites of CA3 pyramidal neurons of the postnatal hippocampus. To understand the possible function of laminin in axon regeneration within this pathway, we have manipulated laminin synthesis at the mRNA level in a slice culture model of the lesioned mossy system. In this model early postnatal mossy fibers severed near the hilus can regenerate across the lesion and elongate rapidly within strata lucidum and pyramidale. In slice cultures of the postnatal day 4 hippocampus, 2 d before lesion and then continuing for 1-5 d after lesion, translation of the gamma1 chain product of laminin was reduced by using antisense oligodeoxyribonucleotides and DNA enzymes. In the setting of the lesioned organotypic hippocampal slice, astroglial repair of the lesion and overall glial patterning were unperturbed by the antisense or DNA enzyme treatments. However, unlike controls, in the treated, lesioned slices the vast majority of regenerating mossy fibers could not cross the lesion site; those that did were very much shorter than usual, and they took a meandering course. In a recovery experiment in which the DNA enzyme or antisense oligos were washed away, laminin immunoreactivity returned and mossy fiber regeneration resumed. These results demonstrate the critical role of laminin(s) in an axon regeneration model of the CNS.