Victor Nurcombe

Association and release of the amyloid protein precursor of Alzheimer's disease from chick brain extracellular matrix

The amyloid protein precursor (APP) of Alzheimers disease was found to bind saturably (Kd = 60 nM) to embryonic chick brain extracellular matrix (ECM). The binding of APP to ECM was not inhibited by 10 micrograms/ml heparin or heparan sulfate. However, pretreatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, reduced the number of APP binding sites on the ECM by 80%. The binding of APP to ECM was also inhibited by pretreatment with chlorate, an inhibitor of glycan sulfation, and heparitinase, which digests the carbohydrate component of heparan sulfate proteoglycans. These results suggest that APP binds with high affinity to one or more heparan sulfate proteoglycans. Acidic and basic fibroblasts growth factor (FGF) also bound to chick ECM. When ECM was incubated with a protease associated with the enzyme AChE (AChE-AP), APP and acidic FGF were released intact from the matrix. The AChE-AP was at least 100-fold more potent in releasing APP from ECM than other trypsin-like proteases (trypsin, plasmin, thrombin). The action of the AChE-AP was inhibited by glia-derived nexin (protease nexin I) and by human brain APP at low nanomolar concentrations. These results suggest that in vivo an AChE-AP may cleave ECM proteins to regulate the availability of soluble APP or other factors bound to the ECM.

Role of Proteoglycans in Neural Development, Regeneration, and the Aging Brain

Proteoglycans (PGs) are a diverse group of macromolecules found in the extracellular matrix (ECM) and on the surface of cells. They have been shown to be coreceptors for several growth factors and are now known to play a crucial role in the regulation of cellular differentiation, process outgrowth, and synaptogenesis. Disturbances in PG metabolism may influence wound repair and may underlie certain pathological features of Alzheimer’s disease.

Expression and localization of FGF-1 in the developing rat olfactory system

Primary olfactory axons project from the nasal olfactory neuroepithelium to glomeruli in the olfactory bulb where they synapse with mitral cells, the second‐order olfactory neurons. We have shown that the heparin‐binding growth factor FGF‐1 is expressed by olfactory nerve ensheathing cells which surround fascicles of primary olfactory axons en route to the olfactory bulb. These cells are believed to modulate olfactory axon growth between the olfactory neuroepithelium and the olfactory bulb. During late embryogenesis, FGF‐1 expression is turned on in the mitral cells, and the FGF‐1 peptide becomes confined to layers of synaptic neuropil in the postnatal olfactory bulb. FGF‐1 is selectively present in glomeruli and the external plexiform layer. In cultures of olfactory neuroepithelial cells, complexes between FGF‐1 and an appropriate activating heparan sulfate proteoglycan stimulated morphological differentiation of both olfactory nerve ensheathing cells and primary sensory olfactory neurons. Thus, the spatiotemporal expression and the functional properties of FGF‐1 in this system suggest that this molecule plays an important regulatory role in the formation of the olfactory pathway.

Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease

Abstract: The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite‐promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin‐binding domain of APP. A fraction 17‐fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity‐purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high‐ and low‐affinity sites with equilibrium dissociation constants of 1.4 × 10−11 and 6.5 × 10−10M, respectively. APP, in conjunction with the affinity‐purified proteoglycan fraction, promoted neurite outgrowth. The affinity‐purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity‐purified fraction with heparitinase I revealed a core protein of 63–69‐kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP‐binding proteoglycans may be an important step in the regulation of the neurite outgrowth‐promoting activity of APP.

The Role of Heparan Sulfate Proteoglycans in the Pathogenesis of Alzheimer's Diseasea

The hallmark of Alzheimers disease (AD) is the deposition of amyloid plaques and neurofibrillary tangles in the brain. The relationship between amyloid deposition and the cognitive deficit is still unclear. The amyloid βA4 protein is produced by proteolytic cleavage of the amyloid protein precursor (APP). Very little is known about the normal function of APP and the role the protein may play in pathogenesis. Several studies have shown that APP is important for the regulation of neurite outgrowth. Our studies support these findings and indicate that the neurite outgrowth‐promoting effects of APP are stimulated by an interaction between APP and specific proteoglycans. Using site‐directed mutagenesis, a heparan sulfate binding site which mediates this effect has been mapped to the N‐terminus of APP (residues 96‐110, HBD‐1). A peptide homologous to HBD‐1 blocks the trophic effects of APP in cell culture. To purify specific proteoglycans which stimulate the action of APP, an affinity column was constructed using a biotinylated peptide homologous to HBD‐1 coupled to streptavidin‐agarose. Two proteoglycans were isolated from a crude brain cell conditioned medium by affinity chromatography. The purified proteoglycans bound APP saturably with high affinity and stimulated the action of APP on neurite outgrowth from chick sympathetic neurons. Digestion of the proteoglycan fraction with heparitinase I or chondroitinase ABC demonstrated the presence of two major proteins, a heparan sulfate proteoglycan with a core protein of 63‐67 kD molecular mass and a chondroitin sulfate proteoglycan with a core protein of 100‐110 kD molecular mass. The results demonstrate that APP binds to at least two proteoglycans and that this interaction may regulate the trophic effects of the protein. The interaction of specific APP‐binding proteoglycans with amyloid plaques may disturb the normal function of APP and contribute to the neuritic degeneration that is commonly seen around the amyloid plaque cores.

Light-microscopic immunolocalization of fibroblast growth factor-1 and -2 in adult rat kidney

Abstract.The fibroblast growth factors (FGFs) are a family of conserved polypeptides known to regulate cell differentiation and proliferation. We have used avidin-biotin-enhanced indirect immunohistochemistry to localize FGF-1 and FGF-2 in the rat kidney. The most consistent specific immunostaining pattern is found in paraffin sections from kidneys perfusion-fixed with 4% paraformaldehyde in 0.1u2005M phosphate buffer. Intracellular immunoreactivity for FGF-1 and FGF-2 is co-localized in visceral (podocytes) and parietal (Bowman’s capsule) glomerular epithelial cells, S3 segments of proximal tubules, distal tubules and collecting ducts in the cortex, and thick ascending limbs and collecting ducts in the medulla. Immunoreactivity is also observed within urothelium and the tunica adventitia of large blood vessels. No immunostaining is found in cortical S1 or S2 segments of proximal tubules, in frozen sections prepared from unfixed or 4% paraformaldehyde perfusion-fixed kidneys, or in paraffin sections from Bouin-fixed kidneys. Immersion fixation with 4% paraformaldehyde gives a similar staining pattern in paraffin sections to that achieved with perfusion fixation. However, in paraffin sections fixed with methyl Carnoy’s fixative, immunoreactivity is primarily localized to the tunica media of blood vessels, with little tubular or glomerular immunostaining. Thus, variation in immunolocalization patterns for FGFs can be partially attributed to differences in fixative, preparative technique and antibody specificity.

Laminin in neural development

This short and selective review of the role of laminin in neural development discusses emerging concepts about the way that elements of the extracellular matrix control the differentiation of embryonic neurons. New laminin isoforms have recently been discovered, discoveries which now reveal the very great heterology of basement membranes in different regions of the nervous system, at different stages of development. The problems of identifying true, neuronal-specific laminin receptors are also discussed, particularly with reference to neuronal pathway formation.

Immunolocalization of fibroblast growth factor-1 and -2 in the embryonic rat kidney

Summary: Fibroblast growth factors (FGF) regulate cell proliferation, migration, differentiation and angiogenesis during morphogenesis in many different tissues. Recent evidence indicates that exogenous FGF‐2 stimulates mesenchymal condensation in cultured rat metanephroi, a crucial epithelial‐mesenchymal induction event in the developing nephron. the aim of the present investigation was to determine the in vivo distribution of FGF‐1 and FGF‐2 in developing rat metanephroi at embryonic days 14, 15, 16, 18 and 20. Avidin‐biotin enhanced indirect immunohistochemistry was used to demonstrate that both FGF‐1 and FGF‐2 were co‐localized in metanephroi at all ages studied. High levels of FGF‐1 and FGF‐2 were present in ureteric bud branches and in developing distal tubules. Fibroblast growth factor‐1 and FGF‐2 were colocalized in developing nephron elements, from vesicles to S‐shaped bodies, and in the mesangium of capillary loop and maturing stage glomeruli. Both growth factors were present in the mesenchyme of the nephrogenic zone and in the interstitium of the developing cortex. However, immunostaining for FGF was not evident in mesenchymal condensates, endothelial cells, medullary interstitial cells, or in the thin undifferentiated epithelium of the immature loop of Henle. These findings indicate that the expression of both FGF‐1 and FGF‐2 is tightly regulated in the embryonic kidney and suggest a role for these molecules in kidney development.

Expression of transforming growth factor-β type II receptor mRNA in embryonic and adult rat kidney

Summary: The transforming growth factor‐β (TGF‐β) family of growth factors regulates cell proliferation, differentiation, extracellular matrix synthesis and angiogenesis in many developing tissues. Transforming growth factor‐β1 was recently shown to affect the branching of ureteric epithelium and nephron formation in cultured rat metanephroi. As the TGF‐β type II receptor is specific for the TGF‐β family, the present study used in situ hybridization to localize mRNA for this receptor in metanephroi from Sprague‐Dawley rat embryos. Transforming growth factor‐β type II receptor mRNA was located in ureteric duct epithelium, undifferentiated mesenchymal cells in the nephrogenic zone, vesicles, comma‐shaped bodies and S‐shaped bodies. In some S‐shaped bodies, TGF‐β type II receptor mRNA was not expressed in the lower limb, which subsequently forms the renal corpuscle. Expression was not observed in capillary loop stage glomeruli and maturing glomeruli, or in proximal tubules and interstitial cells. In adult rat kidney, TGF‐β type II receptor mRNA was expressed in cortical collecting ducts and distal tubules but not in glomeruli or proximal tubules. These findings demonstrate that the prominent expression of TGF‐β type II receptor mRNA decreases as glomeruli and tubules develop. Expression then remains undetectable in adult glomeruli and proximal tubules. the developmentally‐regulated expression of this receptor suggests a key role in glomerular and nephron development.

Chapter 3 – Laminin in neural development

Publisher Summary nThis chapter describes the role of laminin in neural development. Laminin belongs to a supergene family of proteins with genetically distinct subunit chains that associate into many, generally trimeric isoforms. Laminin potentiates the differentiation of embryonal carcinoma into neurons, strongly indicating that specific interactions between an early precursor cell population and extracellular laminin are required during early neural differentiation. Cells with various integrin-like receptors for laminin are attached equally well to laminin-2 and laminin-1, suggesting that several of the known laminin-binding receptors also bind to laminin-2. Antibodies to the β-l subunit of the integrin receptor family inhibit the neurite outgrowth stimulated by laminin-2, confirming the involvement of integrin-mediated interaction of cells with both laminin-1 and -2. The authentic neuronal cell surface receptor binds to a functional site on the laminin molecule formed by the folding of some or all three chains. The laminin-3 homologue concentrated in synaptic clefts of the neuromuscular junction is reported to contain a crucial tripeptide, LRE (leucine-arginine-glutamate), which mediates binding to neuronal cells.