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The extracellular matrix protein Agrin has been detected in chondrocytes and endosteal osteoblasts but its function in osteoblast differentiation has not been investigated yet. Thus, it is possible that Agrin contributes to osteoblast differentiation and, due to Agrin and wingless-related integration site (Wnt) sharing the same receptor, transmembrane low-density lipoprotein receptor-related protein 4 (Lrp4), and the crosstalk between Wnt and bone morphogenetic protein (BMP) signalling, both pathways could be involved in this Agrin-mediated osteoblast differentiation. Confirming this, Agrin and its receptors Lrp4 and α-dystroglycan (Dag1) were expressed during differentiation of osteoblasts from three different sources. Moreover, the disruption of Agrin impaired the expression of its receptors and osteoblast differentiation, and the treatment with recombinant Agrin slightly increase this process. In addition, whilst Agrin knockdown downregulated the expression of genes related to Wnt and BMP signalling pathways, the addition of Agrin had no effect on these genes. Altogether, these data uncover the contribution of Agrin to osteoblast differentiation and suggest that, at least in part, an Agrin-Wnt-BMP circuit is involved in this process. This makes Agrin a candidate as target for developing new therapeutic strategies to treat bone-related diseases and injuries., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with β-dystroglycan (β-DG). Conversely, agrin sliencing profoundly disrupts β-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and β-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin., (© 2019 Wiley Periodicals, Inc.)

The clinical consequences of arterial atherosclerotic lesions depend, apart from their size, on their composition of cellular and extracellular components. While an intact endothelium at the interface of atherosclerotic plaques towards the blood can prevent its erosion, underlying smooth muscle cells within the plaque can reduce the risk of plaque ruptures, due to the deposition of stabilizing extracellular matrix. Basement membranes underlay and support the function of endothelial cells, and embed smooth muscle cells in the media, the source of most smooth muscle cells within atherosclerotic plaques. In the present study mouse atherosclerotic plaques were comparatively analyzed for the basement membrane components laminin, type IV collagen, perlecan, and agrin. Distinct agrin immunofluorescence was found in the peri-luminal area in mouse carotid atherosclerotic plaques. Agrin was also clearly present in the media, with a significant increase in regions directly associated with plaque tissue. In addition, ten human endarterectomy specimens were investigated for this heparan sulfate proteoglycan. No statistically significant differences in agrin immunofluorescence were noticed between five specimens from symptomatic and five from asymptomatic patients. In all these plaques agrin was present in a distinctive manner in a narrow zone partially or almost completely surrounding the lumen. Additionally it was also present around the small lumina of the CD31-positive neovessels. The presence of agrin at locations with particular importance for the growth and stability of atherosclerotic plaques renders this molecule strategically positioned to influence plaque development and vulnerability.

Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes., (Copyright © 2017 the American Physiological Society.)

As the primary protective barrier for neurons in the brain, the blood-brain barrier (BBB) exists between the blood microcirculation system and the brain parenchyma. The normal BBB integrity is essential in protecting the brain from systemic toxins and maintaining the necessary level of nutrients and ions for neuronal function. This integrity is mediated by structural BBB components, such as tight junction proteins, integrins, annexins, and agrin, of a multicellular system including endothelial cells, astrocytes, pericytes, etc. BBB dysfunction is a significant contributor to the pathogeneses of a variety of brain disorders. Many signaling factors have been identified to be able to control BBB permeability through regulating the structural components. Among those signaling factors are inflammatory mediators, free radicals, vascular endothelial growth factor, matrix metalloproteinases, microRNAs, etc. In this review, we provide a summary of recent progress regarding these structural components and signaling factors, relating to their roles in various brain disorders. Attention is also devoted to recent research regarding impact of pharmacological agents such as isoflurane on BBB permeability and how iron ion passes across BBB. Hopefully, a better understanding of the factors controlling BBB permeability helps develop novel pharmacological interventions of BBB hyperpermeability under pathological conditions.

Proteins in living organisms have names that are usually derived from their function in the biochemical system their discoverer was investigating. Typical examples are acetylcholinesterase and agrin; however, for both of these, various other functions that are not related to the cholinergic system have been revealed. Our investigations have been focused on the alternative roles of acetylcholinesterase and agrin in the processes of muscle development and regeneration. Previously, we described a role for agrin in the development of excitability in muscle contraction. In this study, we report the effects of agrin on secretion of interleukin 6 in developing human muscle. At the myoblast stage, agrin increases interleukin 6 secretion. This effect seems to be general as it was observed in all of the cell models analysed (human, mouse, cell lines). After fusion of myoblasts into myotubes, the effects of agrin are no longer evident, although agrin has further effects at the innervation stage, at least in in vitro innervated human muscle. These effects of agrin are another demonstration of its non-synaptic roles that are apparently developmental-stage specific. Our data support the view that acetylcholinesterase and agrin participate in various processes during development of skeletal muscle.

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Objective: To explore a time-efficient method of identifying motor and sensory fascicles in peripheral nerve trunk., Methods: Thirty Wistar rats were selected to obtain whole spine. The spinal dorsal roots and ventral roots, and sciatic nerve were harvested as sensor, motor, and mixed samples, annexin V and agrin specificities were observed with Western blot and immunohistochemistry. A total of 32 New Zealand rabbits were selected and killed. The roots of spinal nerves were exposed under an operating microscope, and the ventral and dorsal roots, ∼3 mm to 5 mm, were dissociated, and frozen as transverse sections of 30-μm thickness. The sections were examined by micro-Raman spectroscopy., Results: The annexin V and agrin were special substances of sensory and motor nerves, respectively, and can act as specific antigens for identifying different nerve fascicles. Sections of the same type of nerve fascicles showed reproducibility with similar spectral features. Significant differences in the spectral properties, such as the intensity and breadth of the peak, were found between motor and sensory fascicles in the frequency regions of 1,088 cm(-1), 1,276 cm(-1), 1,439 cm(-1), 1,579 cm(-1), and 1,659 cm(-1). With the peak intensity ratio of 1.06 (I(1276)/I(1439)) as a standard, we could identify motor fascicles with a sensitivity of 88%, specificity of 94%, positive predictive value of 93%, and negative predictive value of 88%. In the range of 2,700 cm(-1) to 3,500 cm(-1), the half-peak width of the motor fascicles was narrow and sharp, whereas that of the sensory fascicles was relatively wider. A total of 91% of the peak features were in accordance with the identification standard., Conclusion: Motor and sensory fascicles exhibit different characteristics in Raman spectra, which are constant and reliable. Therefore, it is more effective than immunohistochemistry method in identifying different nerve fascicles according to the specific spectrum, and it possesses feasibility for clinical application.

In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs α-β-dystroglycan (DG), α-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/α-β-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of α-β-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane.

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained >10 and >200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics., (Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and is the fifth most common malignancy worldwide. HCC typically develops in the cirrhotic liver. Our preliminary results indicated that agrin, a heparan sulfate proteoglycan (HSPG) detected by us for the first time in the liver, accumulates in the basement membranes (BMs) of the cirrhotic liver and HCC. This novel finding prompted us to investigate the role of agrin in the pathogenesis and differential diagnosis of HCC. First, the previously unspecified monoclonal antibody anti-HSPG clone 7E12 was verified as anti-agrin, using mass spectrometry. Our subsequent experiments were carried out on specimens from 131 patients with chronic liver disease and 18 individuals with healthy liver, from 4 rats subjected to cirrhosis/HCC induction and 1 untreated control rat, as well as from cultured cells. In both human and rats, significantly increased expression of agrin in cirrhosis and HCC was demonstrated by immunohistochemistry (IHC), Western blot, and quantitative RT-PCR. By double immunofluorescent studies, agrin was localized to the muscular layer of blood vessel walls, the BM of bile ducts and ductular reaction, the microvessel walls of HCC, and occasionally the BM of hepatocellular tumor cells. Colocalization, gene expression, and mRNA in situ hybridization experiments suggested that the sources of agrin include vascular smooth muscle cells, epithelial cells of bile ducts and ductules, activated mesenchymal cells in the stroma of hepatocellular tumors, and occasionally tumor hepatocytes. Agrin in the BMs of bile ducts and blood vessels is thought to play an important role in the survival of bile duct epithelium and vascular endothelium, respectively. Thus, agrin may contribute to the formation of ductular reaction and HCC neovessels. As opposed to HCC neovessels that were consistently found agrin-positive, normal and cirrhotic sinusoids were always devoid of agrin, raising the possibility that agrin IHC might be useful in the differential diagnosis of benign versus malignant hepatocellular lesions. Agrin IHC was performed on 68 benign lesions (8 large regenerative nodules, 23 low-grade and 7 high-grade dysplastic nodules, 30 liver adenomas) and 29 malignant lesions (8 small HCC, 21 HCC), and was evaluated semi-quantitatively. Based on the results of IHC for agrin as well as CD34, a decision algorithm was devised that differentiated benign and malignant parenchymal lesions with a sensitivity of 93.1% and a specificity of 92.6%. Hence, we propose that agrin IHC might help distinguish between malignant hepatocellular lesions and their benign mimickers.

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive-motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive-motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of alpha 7B-integrin, beta 1D-integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of alpha 7A-integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of alpha 7B- and beta 1D-integrin mRNA and a normal, or slightly higher than normal, level of alpha 7A-integrin mRNA in the skeletal muscle fibres of the patients with sensitive-motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with alpha 7B-integrin being replaced by alpha 7A-integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down-regulation of integrins and agrin in muscular dystrophies.

Objective: To explore a method to identify the sensory and motor fascicles in peripheral nerve trunk., Methods: Thirty Wistar rats were selected to obtain whole spine. The spinal ganglion, its dorsal root and ventral root, and sciatic nerve were harvested, Annexin V and Agrin specificities were observed with Western blot. In the experimental group, anterior branch and posterior branch of spinal nerve, sciatic nerve, and its muscular branch and cutaneous branch were harvested from 15 rats to make the observation of immunohistochemistry. In the other 15 rats, first antibody was replaced by PBS as control group. Different nerve fascicles were studied with Micro Raman scattering technique in 16 12-month-old New Zealand rabbits., Results: The Annexin V and Agrin were special substances of sensory and motor nerves respectively and can act as specific antigens for identifying different nerve fascicles. There were significant differences in the intensity and breadth of the peak of the spectral properties between motor and sensory fascicles at frequencies of 1,088, 1,276, 1,439, 1,579 and 1,659 cm(-1). The peak intensity ratios of 1,276 to 1,439 cm(-1) were 0.95+/-0.06 in motor nerve fascicles and 1.17+/-0.08 in sensory fascicles, showing significant differences (P<0.05)., Conclusion: The Micro Raman spectra is more effective than immunohistochemistry in identifying different nerve fascicles, and it possesses as feasibility for clinical application.

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as alphav-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.

Factor H, a regulatory protein of the alternative pathway of complement (APC), is present in amyloid-beta (Abeta) plaques in Alzheimer's disease (AD). Abeta plaques also contain significant amounts of heparan sulfate proteoglycans (HSPGs), such as agrin, as well as numerous activated microglia expressing increased levels complement receptor 3 (CR3). Here, we show the colocalization of each of these molecules in the AD brain and the functional capacity for these molecules to bind to one another in vitro. We propose that CR3 receptors expressed by microglia are used for ligand binding to factor H bound to HSPGs and Abeta in plaques in the AD brain.

The quality of the blood-brain barrier (BBB), represented mainly by endothelial tight junctions (TJ), is now believed to be dependent on the brain microenvironment and influenced by the basal lamina of the microvessels. In the highly vascularized glioblastoma multiforme (GBM), a dramatic increase in the permeability of blood vessels is observed but the nature of basal lamina involvement remains to be determined. Agrin, a heparan sulfate proteoglycan, is a component of the basal lamina of BBB microvessels, and growing evidence suggests that it may be important for the maintenance of the BBB. In the present study, we provide first evidence that agrin is absent from basal lamina of tumor vessels if the TJ molecules occludin, claudin-5 and claudin-1 were lacking in the endothelial cells. If agrin was expressed, occludin was always localized at the TJ, claudin-5 was frequently detected, whereas claudin-1 was absent from almost all vessels. Furthermore, despite a high variability of vascular phenotypes, the loss of agrin strongly correlated with the expression of tenascin, an extracellular matrix molecule which has been described previously to be absent in mature non-pathological brain tissue and to accumulate in the basal lamina of tumor vessels. These results support the view that in human GBM, BBB breakdown is reflected by the changes of the molecular compositions of both the endothelial TJ and the basal lamina.

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Galbeta1,3GalNAc and Galbeta1,4GIcNAc are the subterminal saccharide structures present on the CT carbohydrate antigen GalNAcbeta1,4[NeuAcalpha2,3]-Galbeta1-(3GalNAc or 4GIcNAc)-R, which is localized at the mammalian neuromuscular junction. Here we show that Galbeta1,3GalNAc, Galbeta1,4GIcNAc, and the CT carbohydrate antigen affect postsynaptic assembly in cultured muscle cells. Treatment of C2C12 myotubes with benzyl-O-alpha-GalNAc or neuraminidase increased peanut agglutinin (PNA) expression and AChR clustering. Induction of AChR clustering was blocked by PNA and by muscle agrin. Addition of Galbeta1,4GIcNAc or Galbeta1,3GalNAc increased AChR clustering in myotubes and muscle-specific kinase (MUSK) autophosphorylation in vitro, while NeuAcalpha2,3Galbeta1,4GIcNAc and Galbeta1,4GIc did not. Neural agrin activated MuSK in vitro if the lactosamine-containing mucin domain was present, and this activation was blocked in large part by Galbeta1,3GalNAc and Galbeta1,4GIcNAc. Agrin fragments and MuSK bound to these disaccharides with differing specificities. Overexpression of the CT carbohydrate antigen also increased AChR clustering and MuSK autophosphorylation in the presence of neural agrin. These data suggest a model in which different portions of the CT carbohydrate structure contribute to agrin-dependent signal transduction.

Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimer's disease brain. Agrin protein binds the amyloidogenic peptide Abeta (1-40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Abeta fibril formation and protect Abeta (1-40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrin's presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Abeta peptide aggregation and/or its persistence in Alzheimer's disease brain., (Copyright 2000 Academic Press.)

The frog neuromuscular junction offers a unique structure in which to observe fine details in the relationship between the motor neuron, muscle, and Schwann cell, which together comprise the neuromuscular junction. Schwann cell processes that extended from the synapse stained positively with anti-agrin antibodies. Immunocytochemistry revealed strong anti-agrin staining of the extracellular matrix surrounding the entire Schwann cell and the Schwann cell processes come in close contact with the muscle fiber. Dual-labeling experiments revealed a lack of acetylcholine receptor (AChR) aggregates on the surface of the muscle fiber directly under these anti-agrin-staining Schwann cell processes. The cDNA that codes for the C-terminal portion of agrin in frog (Rana pipiens) was cloned and sequenced. Polymerase chain reaction of frog brain, nerve, and muscle cDNA revealed that like other species, agrin transcripts that contain the B8, B11, or B19 inserts were observed only in brain tissue. Unlike other species, the exact site of the B inserts in frog was slightly altered, with the B insertion site occurring within a codon sequence. Our data are consistent with the hypothesis that Schwann cells produce agrin which lacks inserts at the B region, and that agrin lacking B inserts does not direct the aggregation of AChRs in vivo under physiological conditions and concentrations., (Copyright 1999 John Wiley & Sons, Inc.)

Mutations in the dystrophin-glycoprotein complex cause muscle degeneration and dysfunctions in the central nervous system, including an impaired synaptic transmission in the outer plexiform layer (OPL) of the retina. To investigate the basis for this ocular phenotype, we analyzed the distribution of beta-dystroglycan, a central member of the dystrophin-glycoprotein complex, in the chick retina by using the 43DAG/8D5 monoclonal antibody. This antibody reacted specifically with chick beta-dystroglycan, as indicated by its staining of the neuromuscular junction, and its reactivity with a single 43-kilodalton band in Western blots. In the retina, beta-dystroglycan was highly concentrated in the OPL and at the vitreal border of the retina, around the inner limiting membrane. Mechanically isolated and flat-mounted inner limiting membranes were stained by the anti-beta-dystroglycan antibody, and this immunoreactivity could be extracted by detergent, indicating that beta-dystroglycan is associated with membranous structures bound to the basal lamina. Consistently, electron microscopy showed a concentration of beta-dystroglycan in the endfeet of Müller glial cells exclusively in the region of direct contact to the inner limiting membrane. In the OPL, beta-dystroglycan immunoreactivity was concentrated in the distal extensions of rod and cone terminals protruding into the outer plexiform layer. There, beta-dystroglycan codistributed with the alpha1beta subunit of the N-type voltage-gated calcium channel. By contrast to previous reports, we did not detect beta-dystroglycan directly associated with the synaptic regions of conventional or ribbon synapses of the retina. These results show that in the retina beta-dystroglycan is exclusively expressed by photoreceptors and glial cells and that beta-dystroglycan is highly concentrated in subcellular regions of glial cell endfeet and photoreceptor terminals. Moreover, the colocalization of beta-dystroglycan with N-type calcium channels in the outer plexiform layer indicates that both proteins might be part of a macromolecular complex.

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs.

At the developing and regenerating neuromuscular junction, agrin is responsible for the formation of aggregates containing the acetylcholine receptor (AChR) and other molecules. Multiple isoforms of agrin are generated by alternative splicing, and the presence of an 8, 11, or 19 (8 + 11) amino acid insert at splice site B is required for agrin's AChR aggregation activity. An antiserum was generated against the 19 amino acid peptide which reacted specifically with the B11 and B19 agrin isoforms. The antiserum blocked the formation of agrin-induced AChR aggregates on myotubes, but the peptide itself had no aggregation activity, suggesting that agrin's active site is close to the splice site, but not the peptide itself. In the embryonic and adult retina anti-peptide immunoreactivity was concentrated in the synapse-containing layers. In contrast, the inner limiting membrane and radial cells, which express strong immunoreactivity with a pan-specific anti-agrin antiserum, were not stained by the anti-peptide antiserum, showing that agrin isoforms are differentially distributed in the retina. In addition, agrin B11 and B19 isoforms were associated with ganglion cell axons, particular at early developmental stages before synapse formation, indicating additional functions for these isoforms in the developing CNS.

The blood-brain barrier (BBB) is an essential feature of the microvasculature in neural tissues. Agrin, a synapse organizing molecule at the neuromuscular junction, also accumulates on brain microvasculature and may be involved in BBB formation and function. We investigated the developmental expression of agrin at rat and chick brain capillaries by immunohistochemistry and immunoblotting. Anti-agrin immunoreactivity (IR) completely ensheathes all observed microvessels labeled with anti-von Willebrand factor in adult brains of both species. Brain microvascular agrin IR codistributes with anti-laminin IR, consistent with agrin localization in the microvessel basal lamina. On microvessels in testis and thymus, tissues which also contain blood-tissue barriers, the pattern of IR is indistinguishable from brain microvessels. In contrast, little or no agrin IR is observed on capillaries in muscle and other tissues. During chick and rat development, agrin accumulates on brain microvessels around the time the vasculature becomes impermeable. Differential staining and electroblotting suggest that the agrin isoforms expressed on brain microvessels lack the 8- and 11-amino acid sequences that confer on agrin high potency in acetylcholine receptor clustering. Taken in context with the organizing role of agrin in synaptogenesis, these results indicate that agrins may function as important players in the formation and maintenance of cerebral microvascular impermeability.

Agrin, an extracellular matrix protein synthesized by nerves and muscles is known to promote the clustering of acetylcholine receptors and other synaptic proteins in cultured myotubes. This observation suggests that agrin may provide at least part of the signal for synaptic specialization in vivo. The extracellular matrix components agrin, laminin and merosin bind to alpha-dystroglycan, a heavily glycosylated peripheral protein part of the dystrophin-glycoprotein complex, previously characterized in the sarcolemma of skeletal and cardiac muscles and at the neuromuscular junction. In order to understand further the function of agrin and alpha DG in the genesis of the acetylcholine receptor-rich membrane domain, the settling of components of the dystrophin-glycoprotein complex and agrin was followed by immunofluorescence localization in developing Torpedo marmorata electrocytes. In 40-45 mm Torpedo embryos, a stage of development at which the electrocytes exhibit a definite structural polarity, dystrophin, alpha/beta-dystroglycan and agrin accumulated concomitantly with acetylcholine receptors at the ventral pole of the cells. Among these components, agrin appeared as the most intensely concentrated and sharply localized. The scarcity of the nerve-electrocyte synaptic contacts at this stage of development, monitored by antibodies against synaptic vesicles, further indicates that before innervation, the machinery for acetylcholine receptor clustering is provided by electrocyte-derived agrin rather than by neural agrin. These observations suggest a two-step process of acetylcholine receptor clustering involving: (i) an instructive role of electrocyte-derived agrin in the formation of a dystrophin-based membrane scaffold upon which acetylcholine receptor molecules would accumulate according to a diffusion trap model; and (ii) a maturation and/or stabilization step controlled by neural agrin. In the light of these data, the existence of more than one agrin receptor is postulated to account for the action of agrin variants at different stages of the differentiation of the postsynaptic membrane in Torpedo electrocytes.

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs, must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and alpha-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction.

In order to disclose the mechanism that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo, agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.