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Are heparan sulphate (HS) sulphotransferases implicated in the pathogenesis of Alzheimer's disease?
... A 'consensus sequence' for heparin/HS binding (Cardin and Weintraub, 1989) has been identified corresponding to residues 12-17 of the A, peptide, and interaction with the sulphate substituents of GAG chains is believed to influence the aggregation behaviour of the peptide (Fraser et al., 1992). Indeed, it has been speculated that altered binding properties due to deranged sulphation of HS may be a primary cause of the disease (Zebrower and Kieras, 1993). However, little is known regarding the structure of HS GAG from human brain, let alone the correlation to AD. ...
... Functional abberations might be considered in relation to the slight elevation in Nsulphation noted for some of the AD cerebral HS preparations in the present study. However, the findings do not support the proposal of a primary structural change in the HS molecule, caused by altered control of O-sulphotransferases in HS biosynthesis (Zebrower and Kieras, 1993), as a causative factor in AD. On the other hand, it seems likely that overproduction of the amyloid fl-protein, for whatever reason, may lead to the formation of ,-protein-HS complexes, with secondary effects on vital HS functions. ...
Heparan sulphate (HS) was isolated after proteolytic digestion of cerebral cortex, obtained at autopsy, of patients with Alzheimer's disease (AD) and of control subjects. Deaminative cleavage in combination with selective radiolabelling procedures showed that the N-acetylated regions in the intact polysaccharides ranged from isolated residues to approximately 10 consecutive N-acetylated disaccharide units, without any apparent difference between AD and control HS. The yield of disaccharide deamination products was slightly higher with AD than with control HS, suggesting a differential distribution of N-sulphate groups. Separation of the disaccharides by anion-exchange h.p.l.c. yielded four mono-O-sulphated and one di-O-sulphated disaccharide; these components occurred in strikingly similar proportions in all cerebral HS preparations (except polysaccharide from neonatal brain) irrespective of the age of the individual and the histopathology of the cortex specimen. No significant difference was noted between HS obtained from control and from AD tissue. By contrast, the composition of HS isolated from brain differed significantly from that of HS preparations derived from other human organs.
... Zebrower and Kieras reported that cultured skin fibroblasts from AD patients produced 30% more GlcA-GlcNSO3, and 40% less GlcA-GlcN-6-OSO3 compared to samples from agematched healthy individuals (Zebrower et al., 1992). Authors suggested that an alteration in HS-sulfotransferase activity was responsible for their observations (Zebrower and Kieras, 1993). Importantly, a critical requirement for the presence of 6-O-sulfated disaccharide-containing HS in internalization and spreading of infectious tau species has recently been demonstrated (Rauch et al., 2018;Stopschinski et al., 2018). ...
Microtubule-associated protein tau (MAPT) hyperphosphorylation and aggregation, are two hallmarks of a family of neurodegenerative disorders collectively referred to as tauopathies. In many tauopathies, including Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and Pick’s disease, tau aggregates are found associated with highly sulfated polysaccharides known as heparan sulfates (HSs). In AD, amyloid beta (Aβ) peptide aggregates associated with HS are also characteristic of disease. Heparin, an HS analog, promotes misfolding, hyperphosphorylation and aggregation of tau protein in vitro. HS also provides cell surface receptors for attachment and uptake of tau seeds, enabling their propagation. These findings point to HS-tau interactions as potential therapeutic targets in tauopathies. The zebrafish genome contains genes paralogous to MAPT, genes orthologous to HS biosynthetic and chain modifier enzymes, and other genes implicated in AD. The nervous system in the zebrafish bears anatomical and chemical similarities to that in humans. These homologies, together with numerous technical advantages, make zebrafish a valuable model for investigating basic mechanisms in tauopathies and identifying therapeutic targets. Here, we comprehensively review current knowledge on the role of HSs in tau pathology and HS-targeting therapeutic approaches. We also discuss novel insights from zebrafish suggesting a role for HS 3-O-sulfated motifs in tau pathology and establishing HS antagonists as potential preventive agents or therapies for tauopathies.
... A specific study addressing β4GalT, GlcAT-P, GlcAT-S, and HNK-1ST enzymatic activities in the AD brain is warranted. Altered sulfotransferase activities have been observed in AD pathogenesis [32,33]. Moreover, other enzymes, such as β-glucuronidase, which can be involved in HNK-1 synthesis [34], have been demonstrated to be altered in AD [35], suggesting that further studies should be done on the levels and activities of other glycosidases and glycosyltransferases in AD. ...
The human natural killer-1 (HNK-1), 3-sulfonated glucuronic acid, is a glycoepitope marker of cell adhesion that participates in cell-cell and cell-extracellular matrix interactions and in neurite growth. Very little is known about the regulation of the HNK-1 glycan in neurodegenerative disease, particularly in Alzheimer's disease (AD). In this study, we investigate changes in the levels of HNK-1 carrier glycoproteins in AD. We demonstrate an overall decrease in HNK-1 immunoreactivity in glycoproteins extracted from the frontal cortex of AD subjects, compared with levels from non-demented controls (NDC). Immunoblotting of ventricular post-mortem and lumbar ante-mortem cerebrospinal fluid with HNK-1 antibodies indicate similar levels of carrier glycoproteins in AD and NDC samples. Decrease in HNK-1 carrier glycoproteins were not paralleled by changes in messenger RNA (mRNA) levels of the enzymes involved in the synthesis of the glycoepitope, β-1,4-galactosyltransferase (β4GalT), glucuronyltransferases GlcAT-P and GlcAT-S, or sulfotransferase HNK-1ST. Over-expression of amyloid precursor protein in Tg2576 transgenic mice and in vitro treatment of SH-SY5Y neuroblastoma cells with the amyloidogenic Aβ42 peptide resulted in a decrease in HNK-1 immunoreactivity levels in brain and cellular extracts, whereas the levels of soluble HNK-1 glycoproteins detected in culture media were not affected by Aβ treatment. HNK-1 levels remain unaffected in the brain extracts of Tg-VLW mice, a model of mutant hyperphosphorylated tau, and in SH-SY5Y cells over-expressing hyperphosphorylated wild-type tau. These results provide evidence that cellular levels of HNK-1 carrier glycoforms are decreased in the brain of AD subjects, probably influenced by the β-amyloid protein.
Increasing evidence exists to support a potentially crucial role for proteoglycans, in particular heparan sulfate proteoglycans,
in the pathophysiology of many protein conformational disorder diseases. This chapter will focus on emerging evidence that
supports a role for proteoglycans in the regulation of protein conformation in amyloid diseases, with the result being that
proteoglycans are attractive therapeutic targets for these diseases. Amyloid diseases in both the nervous system and nonneural
tissues share a common property with proteoglycans, especially heparan sulfate proteoglycans, being associated with amyloid
fibrils coincident with the time amyloid fibrils are formed in the disease process. The binding of heparan sulfate proteoglycans
to the specific amyloidassociated protein has been shown in many cases to lead to conformational changes in the amyloidassociated
protein, with an introduction of β-sheet structure to the amyloid protein. Heparan sulfate proteoglycans have also been demonstrated
to accelerate the formation of amyloid oligomers, protofibrils, and fibrils, as well as impart resistance to proteolytic degradation
of the amyloid fibrils. Heparan sulfate proteoglycans therefore appear to play a critical role in the progression of amyloid
diseases, and an ability to prevent heparan sulfate binding to amyloid-associated proteins may allow both an inhibition of
the formation of new amyloid, as well as promote the clearance of existing amyloid.
Inflammation and the response to injury may play an important role in the process of amyloidosis in Alzheimer's disease. We investigated the effect of interleukin-1 (IL-1) and nerve growth factor (NGF) on the metabolism of neuroblastoma proteoglycans. IL-1 and NGF increased the net charge and the net secretion of neuroblastoma proteoglycans. NGF also specifically increased the relative amount of cell-associated and secreted heparan sulfate proteoglycans in these cells. We previously demonstrated that neuroblastoma heparan sulfate proteoglycan binds specifically to the amyloid beta-amyloid peptide involved in Alzheimer's disease. Heparan sulfate glycosaminoglycans synthesized by IL-1-stimulated cells demonstrated an increased relative binding affinity for the beta-amyloid peptide. Thus, IL-1 and NGF induce the hypersecretion and hypersulfation of neuroblastoma heparan sulfate proteoglycans which bind beta-amyloid. These studies link the process of inflammation and repair with alterations in the metabolism of heparan sulfate proteoglycans and amyloid formation in Alzheimer's disease and other disorders.
Alzheimer's disease is a dementing disorder affecting increasingly large numbers of individuals in the aging population. The characteristic neuropathologic changes of Alzheimer's disease are the deposition of extracellular amyloid plaques, neurons containing neurofibrillary tangles, and neuronal cell loss. The A4 amyloid peptide is the major constituent of senile plaques. In addition to the A4 peptide, senile plaques contain a variety of molecular species, including proteoglycans and inflammatory components. The presence of proteoglycans in the amyloid deposits of Alzheimer's disease and of systemic amyloidoses suggests that these molecules play an active role in the pathogenesis of amyloidosis. However, the molecular mechanisms that lead to the codeposition of amyloid peptide with proteoglycans is still unknown. Recent evidence suggests that the metabolism of proteoglycans is altered in Alzheimer's disease patients. The acute-phase response observed in the brain of patients affected by Alzheimer's disease may be responsible for this effect. In this article, we discuss the role of proteoglycans in Alzheimer's disease, and the possible interactions between factors involved in brain inflammatory mechanisms and proteoglycans in the pathogenesis of Alzheimer's disease.
Egg yolk provided the starting disialylundecasaccharide for a highly efficient chemoenzymatic synthesis of a biantennary complex-type glycopeptide (see picture). Repetitive protection and deprotection steps required in oligosaccharide synthesis were thus avoided, and the desired glycopeptide was rapidly assembled on a solid phase (R = peptide).
Amyloids are complex tissue deposits and each type is identified by one of 22 different proteins or peptides which become re-folded into non-native conformational intermediates and then assemble into fibrils of a highly regular structure. All amyloid deposits also contain apolipoprotein E (apoE) as well as the basement membrane (BM) components, serum amyloid P and heparan sulfate proteoglycans (HSPG), perlecan or agrin. These BM components likely contribute to the overall organization of amyloid fibrils and HSPG has been further implicated in the genesis of amyloid. A growing body of evidence, summarized in this review, suggests that heparan sulfate (HS) promotes fibrillogenesis by associating with the amyloid precursors and inducing the conformational change required for their assembly into fibrils. HS also remains associated with the nascent fibrils contributing to its stability. These activities of HS are likely mediated through specific binding sites on the precursor proteins which appear to have sequence characteristics that are unique to amyloid.
Familial amyloidotic polyneuropathy (FAP) is a fatal disease, belonging to a group of systemic disorders caused by an amyloidogenic transthyretin (TTR) variant. Orthotopic liver transplantation (OLT) eliminates the source of the variant TTR molecule, and is presently the only known curative treatment. A fascinating consequence of this treatment is the possibility of retransplanting the removed FAP liver into another non-FAP patient, which created the so-called domino liver transplantation (DLT) procedure. The Familial Amyloidotic Polyneuropathy World Transplant Registry (FAPWTR) was initiated in 1995, and in 1999 a Domino Liver Transplantation Registry (DLTR) was created. Herein data from these Registries are presented. A total number of 56 centers in 16 countries have performed OLT for FAP, and, today, approximately 65-70 OLTs are performed annually worldwide. During the last decade, a total of 623 patients have undergone 660 OLTs. Patient survival is excellent and comparable to the survival with OLT performed for other chronic liver disorders. Twenty-six centers in 12 countries have reported recipients of DLT grafts and presently 30-35 DLTs are performed annually. The FAPWTR and DLTR have become useful tools in evaluating the potential risks and benefits of these relatively new therapeutic options, in addition to encouraging a rewarding collaboration between centers involved in the management of these patients.
The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.
Double-labeling immunohistochemistry was used to investigate the topographical relationship between beta-amyloid and tau protein epitopes present in cells bearing neurofibrillary tangles found in the hippocampal formation of patients with Alzheimer disease. An antiserum raised against the amino terminus of beta-amyloid stained numerous tangle-bearing cells and other bodies ("extracellular tangles"), but double labeling showed that the beta-amyloid staining is invariably peripheral to that of the tau-positive tangle proper. This and other results suggest that the extracellular amyloid plaques and the intracellular neurofibrillary tangles are biochemically distinct but may result from related pathological events that originate at the level of the nerve cell and lead to its degeneration.
Neurofibrillary tangles (NFTs), a hallmark of Alzheimer disease, are commonly located in perikarya of neurons. In advanced cases of Alzheimer disease, however, NFTs are observed also in the extracellular space. As extracellular NFTs (E-NFTs), and occasionally intracellular NFTs (I-NFTs), are recognized by antibodies to beta-amyloid protein (beta AP), beta AP may be present not only in amyloid deposits but also in paired helical filaments (PHFs), the primary components of NFTs. We compared the antigenic characteristics of I-NFTs and E-NFTs with light- and electron-microscopic immunocytochemistry by using several antibodies to noncontiguous epitopes of the microtubule-associated protein tau and of ubiquitin (Ub) as well as an antiserum to beta AP. At variance with I-NFTs, E-NFTs were made predominantly of straight filaments (SFs), rather than PHFs, that were often separated by astroglial processes and in close association with small beta AP deposits. Occasionally, E-NFTs were made of bundles of amorphous material, which showed no resemblance to SFs, PHFs, or amyloid fibrils. The antigenic changes in E-NFTs suggest that when NFTs become extracellular they lose the N and, possibly, the C termini of tau while maintaining the intermediate region of the molecule; they also lose the N-terminal two-thirds of Ub while the C-terminal conjugation site of Ub is preserved. A small subset of E-NFTs reacted with antibodies to both beta AP and tau. Although in most E-NFTs, the epitopes recognized by tau and Ub antibodies were located in typical PHFs and SFs, the epitopes recognized in this subset of anti-beta AP and anti-tau-positive E-NFTs were located exclusively in the bundles of amorphous material. It is suggested that either beta AP epitopes are present but inaccessible in PHFs and SFs and become exposed after conformational changes occurring in the extracellular space or PHFs and SFs become closely associated with beta AP in the extracellular space.
A monoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha 1----4 glucuronic acid-containing determinant in heparan sulfate (HS) chains of a basement membrane-derived heparan sulfate proteoglycan identified and immunolocalized HS specifically to the amyloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle-bearing neurons in the brains of Alzheimer's and Down's syndrome (DS) patients. Ultrastructural immunohistochemistry demonstrated that HS within neurons of Alzheimer's disease (AD) brain was localized to lipofuscin granules, an aging pigment previously shown also to contain beta-amyloid protein (BAP). Heparan sulfate also was localized to neurite-containing, nonfibrillar 'primitive' plaques that also demonstrated positive BAP immunoreactivity in both AD and DS brains. Antibodies to laminin, fibronectin, and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurons of the hippocampus and amygdala as early as 1 day after birth. Young age-matched controls did not demonstrate similar positive HS immunoreactivity in neurons, whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest deposition of BAP was first observed as 'amorphous' or 'diffuse' cortical deposits in DS brain in patients aged 18 and 24 years before the accumulation of fibrillar amyloid (observed in DS patients who are 35 years and older). These cortical deposits also contained positive HS immunoreactivity, implying that HS accumulation in conjunction with the BAP is an early event that ultimately may contribute to the early age-related accumulation (ie, as early as 35 years of age in DS) of NPs, NFTs, and/or CA. Furthermore the colocalization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may be important in lesion development in these two diseases.
Work from several laboratories has identified a proteoglycan complex secreted by a variety of non-neuronal cells that can promote neurite regeneration when applied to the surface of culture dishes. Using a novel immunization protocol, a monoclonal antibody (INO) was produced that blocks the activity of this outgrowth-promoting factor (Matthew, W. D., and P. H. Patterson, 1983, Cold Spring Harbor Symp. Quant. Biol. 48:625-631). We have used the antibody to analyze the components of the active site and to localize the complex in vivo. INO binding is lost when the complex is dissociated; if its components are selectively reassociated, INO binds only to a complex containing two different molecular weight species. These are likely to be laminin and heparan sulfate proteoglycan, respectively. On frozen sections of adult rat tissues, INO binding is present on the surfaces of glial cells of the peripheral, but not the central, nervous system. INO also binds to the basement membrane surrounding cardiac and skeletal muscle cells, and binding to the latter greatly increases after denervation. In the adrenal gland and kidney, INO selectively reacts with areas rich in basement membranes, staining a subset of structures that are immunoreactive for both laminin and heparan sulfate proteoglycan. In general, the outgrowth-blocking antibody binds to areas known to promote axonal regeneration and is absent from areas known to lack this ability. This suggests that this complex, which is active in culture, may be the physiological substrate supporting nerve regeneration in vivo.
The effects of extracellular matrix components on proteoheparan sulfate biosynthesis was studied for bovine aortic endothelial cells in tissue culture. When the cells were maintained on a variety of different purified components of the extracellular matrix, the cells expressed the same three species of proteoheparan sulfates as the cells cultured on tissue culture plastic (HS I, HS II, and HS III). However, the amounts of the three species recovered from the tissue culture medium were found to be dependent on the substrate on which the cells are grown as well as on other factors. In comparison with plastic, much less HS I was found in the medium of cells maintained on substrates containing diverse matrix molecules, whereas the amounts of HS II and HS III essentially remained the same. In contrast, when bovine aortic organ cultures were analyzed under pulsatile flow, marked differences in the profile of proteoheparan sulfate biosynthesis were observed: HS I was found exclusively associated with the plasma membrane of the endothelial cells; HS II was localized only to the subendothelial matrix; and HS III represented the only proteoheparan sulfate species in the medium. This distribution is consistent with polarized secretion and deposition into the subcellular matrix of HS III and retention of HS I in the plasma membrane in the organ culture situation, a biosynthetic phenotype which can only be approximated at best by maintaining the endothelial cells on a substrate other than plastic. When aortic media (devoid of endothelial cells) was placed in organ culture, no HS III could be detected, which suggested that the vascular endothelial cell is the major cell type responsible for its synthesis in organ culture. Thus, the extracellular matrix, depending upon its composition and organization, may play an important role in stabilizing cell polarity and thereby contribute to maintenance of the differentiated phenotype appropriate for the endothelial cell.
INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.
Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).
The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.
The cytoskeleton of a normal mature neuron is composed of three types of fibrils, the microtubules, the neurofilaments and the microfilaments. One of the cellular and molecular changes with aging, the mechanism of which remains unknown to date, is the formation of argentophilic intracellular neurofibrillary tangles in certain selected neurons of the aged human brain. These extraordinary neurofibrillary changes are seen in great abundance in several adult and late life dementias especially the Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT) (for review see Iqbal, et al. 1977b; Iqbal and Wisniewski 1983). The Alzheimer neurofibrillary tangles (ANT) are composed of paired helical filaments (PHF). Bundles of these PHF are also found in neurites (Braak, et al. 1986), especially in the dystrophic neurites of the neuritic (senile) plaque, the second leading histopathological lesion of AD/SDAT. Together these two lesions, the ANT and the plaques, both of which contain the PHF, are the histopathological hallmark of AD/SDAT (Kidd, 1964; Terry, et al. 1964); occassionally either tangles of 15 nm straight filaments or these filaments admixed with PHF have been observed in a few AD/SDAT cases. (Shibayama and Kitoch, 1978; Yagishita, et. al. 1981). The number of ANT and plaques correlates positively with the degree of psychometric deficiency in the affected patients (Tomlinson, et. al. 1970), but their origin and role in disease are not understood.
A purified protein derived from the twisted beta-pleated sheet fibrils in cerebrovascular amyloidosis associated with Alzheimer's disease has been isolated by Sephadex G-100 column chromatography with 5 M guanidine-HC1 in 1 N acetic acid and by high performance liquid chromatography. Amino acid sequence analysis and a computer search reveals this protein to have no homology with any protein sequenced thus far. This protein may be derived from a unique serum precursor which may provide a diagnostic test for Alzheimer's disease and a means to understand its pathogenesis.
Skin fibroblasts lines established from patients with Alzheimer's disease and old normal individuals were cultured with 35S-sodium sulfate and 3H-glucosamine. Proteoglycans were isolated and characterized. Sulfate incorporation into proteoglycans increased in Alzheimer's disease fibroblasts relative to normal controls. These increases changed the ratio of chondroitin sulfate to heparan sulfate proteoglycan from 1.4 to 1.7 (p = 0.0012) and decreased the ratio of cell to medium proteoglycans from 0.32 to 0.26 in normal and Alzheimer fibroblasts (p = 0.006), respectively. HPLC analysis of the disaccharides produced by chondroitinase ABC revealed no differences in composition between proteoglycans of Alzheimer and normal fibroblasts in either the cell or medium fraction. However, analysis of disaccharides produced by heparinase plus heparitinase showed differences in composition in the medium but not the cell fraction, ΔUA-G1cNS was increased by 30% while ΔUA-G1cNS-6S was reduced by 40% in Alzheimer's disease.
A morphologically differentiated strain of rat pheochromocytoma (PC‐12H) metabolically labeled with [ ³⁵ S]methionine and incubated with a phorbol ester displayed reduced 140‐kDa and increased 15 kDa bands relative to cells incubated without phorbol ester after immunoprecipitation with antisera elicited by the C‐terminal peptide of the Alzheimer amyloid precursor protein (APP). These bands correspond to glycosylated full length APP and a C‐terminal fragment previously reported by Anderson et al. (Neurosci. Lett. 120:126–128, 1991) to result from a cleavage within the amyloidotic A4 region of APP, which releases a 120 kDa extracellular fragment. The 15 kDa fragment, not immunoprecipitated with an antisera elicited by the N‐terminal portion of A4 amyloid, is nonamyloidogenic. Incubation of these cells with p ‐nitrophenylxyloside, known to inhibit proteoglycan formation, also increased this nonamyloidogenic cleavage of APP.
In contrast to these results, an undifferentiated low passage PC‐12‐L strain constitutively displayed rapid nonamyloidogenic APP cleavage. Incubation of PC‐12‐L with phorbol ester did not affect the relative abundance of 140 or 15 kDa bands. Growth of PC‐12‐L with 7 S NGF or dibutyryl cAMP resulted in increased morphological differentiation and deceased APP cleavage which was now phorbol‐inducible. Similar analyses of dividing and senescent human astrocytes and normal and F‐AD fibroblasts indicate 5‐fold lower rates of mid‐A4 APP cleavage. Phorbol esters decreased the 140 kDa APP band without affecting the intensity of the 15 kDa band in these cells. Phorbol esters similarly reduces the 140 kDa band in 1 of 5 human neuroblastomas without affecting a constitutive level of 15 kDa APP which was 10‐fold lower than that seen in PC‐12‐L.
These data indicate that (1) rapid mid‐A4 APP cleavage occurs in additional cell types including CNS astrocytes; (2) nonamyloidogenic APP cleavage is inhibited in constitutively or chemically differentiated PC‐12 cells; (3) phorbol ester inductions of mid‐A4 APP cleavage product accumulations and reduced accumulations of products of APP maturation are not coupled in all cell types; and (4) inhibition of proteoglycan formation increases this APP cleavage. These results may be relevant to alterations in APP processing in AD‐affected brain regions. © 1992 Wiley‐Liss, Inc.
Neurofibrillary tangles (NFT) are abnormal filamentous inclusions that develop in neurons in Alzheimer disease and other disorders. When neurons die, the neurofibrillary tangles that persist in the extracellular space show ultrastructural and antigenic changes. Both intra- and extracellular NFT have recently been shown to contain heparan sulfate proteoglycans (HSPGs). HSPGs are also present in other amyloid deposits in the brain and in systemic amyloidoses. Basic fibroblast growth factor (bFGF) is a heparin binding growth factor which is involved in angiogenesis and also has neurite promoting activity. We now report that bFGF binds avidly to extracellular NFT. Alz-50, a monoclonal antibody (MAb) to an abnormal form of tau and bFGF binding label mutually exclusive subpopulations of neurofibrillary tangles. bFGF binding is abolished by heparinase or heparitinase treatment and therefore is most likely based on the presence of HSPG. Binding of bFGF is a specific and sensitive morphological method to distinguish intra- from extracellular NFT. As intracellular NFT, which also contain HSPGs, are not labeled by bFGF binding, this finding also suggests that HSPGs are modified when the NFT become extracellular.
The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.
Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.
We have investigated the specificities of antibrain antibodies in the sera of patients with senile dementia of the Alzheimer's type (SDAT). Using indirect immunofluorescence, we observed a vascular pattern of staining in 6 of 16 sera from patients with typical SDAT. None of 14 sera from age matched controls demonstrated this pattern of staining. The vascular pattern of staining seen with SDAT sera was identical to the immunofluorescent staining of brain by a monoclonal antibody to vascular basement membrane heparan sulfate proteoglycan. Immunoabsorption of SDAT sera with purified vascular proteoglycan abolished the staining of brain vessels. Using an enzyme linked immunoassay, 3 of the 6 vascular-reactive SDAT sera, and none of the 24 sera from aged controls, were shown to contain antibodies to purified vascular heparan sulfate proteoglycan. Proteoglycans play an important role in the barrier function of the blood-brain barrier. Autoimmune injury to the blood-brain barrier by antivascular antibody may play a role in the pathogenesis of dementia by permitting the passage of injurious substances into the brain.
Glycosaminoglycans (GAGs), localized on the surfaces of cells and in the basement membrane, modulate the growth and differentiation of many cell types. Recent studies have shown that heparin, a GAG found in mast cells, potentiates the ability of acidic fibroblast growth factor (aFGF) to induce neurite outgrowth in pheochromocytoma (PC12) cells. We examined the effect of a variety of GAGs on aFGF, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The effects observed were dependent upon the specific GAG, the concentration of the GAG, and the growth factor. Heparin potentiated aFGF-induced neurite outgrowth in a concentration-dependent fashion; potentiation increased with increasing heparin concentrations of 0.01-100 micrograms/ml. At concentrations greater than 100 micrograms/ml, heparin potentiation decreased. The maximally active concentration of heparin (100 micrograms/ml) increased the potency of aFGF 102-fold. Increasing concentrations of heparan sulfate, dermatan sulfate, and chondroitin sulfate correlated with increasing aFGF potentiation. The maximally active concentrations of heparan sulfate (100 micrograms/ml), dermatan sulfate (10 mg/ml), and chondroitin sulfate (1 mg/ml) increased the activity of aFGF 11-, 110-, and 11-fold, respectively. Hyaluronic acid did not affect the neurite outgrowth-promoting activity of aFGF. Heparin also altered the activity of bFGF; increasing concentrations of heparin (0.01-1 micrograms/ml) correlated with increased potentiation. At concentrations greater than 1 microgram/ml, heparin concentration was inversely correlated with potentiation. Chondroitin sulfate only increased the percentage of neurite-bearing cells at concentrations greater than 10 micrograms/ml. Maximally active concentrations of heparin (1 microgram/ml) and chondroitin sulfate (1 mg/ml) increased the potency of bFGF 5-fold. The highest concentration of heparan sulfate studied (1 mg/ml) inhibited the activity of bFGF. Dermatan sulfate and hyaluronic acid (0.01-1000 micrograms/ml) had no effect on bFGF activity. Heparan sulfate and chondroitin sulfate showed concentration-dependent potentiation of NGF; maximally active concentrations of heparan sulfate (100 micrograms/ml) and chondroitin sulfate (1 mg/ml) increased the potency of NGF 3-fold, whereas heparin, dermatan sulfate and hyaluronic acid had no effect. None of the GAGs had any effect on PC12 neurite outgrowth when added alone. The specificity of the activity of the GAGs was verified by selective enzyme degradation.(ABSTRACT TRUNCATED AT 400 WORDS)
Amyloid is a generic term referring to a group of diseases in which proteinaceous tissue deposits all have in common specific stain affinities, a common appearance in polarized light, common ultrastructure fibrillary characteristics, and uniform x-ray diffraction and infrared spectral properties. Where groups of diseases have a common underlying pathogenetic process the polypeptide responsible for the protein fibril is the same regardless of the specific disease. Where diseases have a different underlying pathogenesis the polypeptide is unique for each disease. The different amyloidogenic polypeptides are clearly not related in terms of amino acid sequence or function, yet they all tend to fold in such a way as to present the same staining, structural or spectral properties. It is proposed that amyloid fibrils are not only composed of the specific amyloidogenic polypeptide but also highly sulphated glycosaminoglycans or proteoglycans which have a profound influence on the manner in which the peptides fold and interact with each other. It is this highly charged carbohydrate which may be common to all amyloids and which plays a determining role in the final appearance of the deposit. Amyloid should therefore be considered as more than simply a protein entity but, as its name originally implied, one related to carbohydrate deposition as well.
Cell-cell interactions are of critical importance during neural development, particularly since the migration of neural cells and the establishment of functional interactions between growing axons and their target cells has been suggested to depend upon cell recognition processes. Neurone-neurone adhesion has been well studied in vitro, and is mediated in part by the neural cell adhesion molecule N-CAM. N-CAM-mediated cell-cell adhesion has been postulated to occur by a homophilic binding mechanism, in which N-CAM on the surface of one cell binds to N-CAM on a neighbouring cell. Studies in our laboratory have identified a cell surface glycoprotein, now known to be N-CAM, which participates in cell-substratum interactions in the developing chicken nervous system. Although this adhesion involves a homophilic binding mechanism, the binding of the cell surface proteoglycan heparan sulphate to the glycoprotein is also required. This raises the question of whether the binding of heparan sulphate to N-CAM is also required for cell-cell adhesion. Here we show that the binding of retinal probe cells to retinal cell monolayers is inhibited by heparin, a functional analogue of heparan sulphate, but not by chondroitin sulphate. Monoclonal antibodies that recognize two different domains on N-CAM, the homophilic-binding and heparin-binding domains, inhibit cell-cell adhesion. The heparin-binding domain isolated from N-CAM by selective proteolysis also inhibits cell-cell adhesion when bound to the probe cells.
Basic fibroblast growth factor (bFGF) is a heparin-binding protein which has trophic effects on hippocampal neurons in vitro. It stimulates neurite extension when bound to surfaces coated with heparin, heparan sulfate, or hyaluronic acid, but not chondroitin sulfate or dermatan sulfate. Stimulation of neurite growth correlated strongly with the amount of [125I]bFGF bound by the different glycosoaminoglycans. Providing accessible stores of bFGF might be one function of glycosoaminoglycans during development.
Cultured skin fibroblasts from four patients with Alzheimer's disease had life spans comparable to those of six age-sex matched controls, whether measured by passages to phase-out, cumulative population doublings to phase out, or percentage of nuclei incorporating [3H]thymidine (Cristofalo index). These results provide direct experimental evidence that Alzheimer's disease is not simply a form of accelerated aging. They suggest that the abnormalities, described by several groups, in Alzheimer fibroblasts reflect the disease rather than the physiological age of the donor, making the cultured cell a valid tool for studying the cellular pathophysiology of this disorder. Together with other data, these observations raise the possibility that some forms of Alzheimer's disease may represent inborn error(s) of metabolism of late clinical onset.
Using the sulfated alcian blue and alcian blue-MgCl2 techniques for demonstrating sulfated glycosaminoglycans (GAGs), we have shown sulfated GAGs to be associated with the amyloidotic lesions of Alzheimer's disease, the neuritic plaques, the neurofibrillary tangle, and the congophilic angiopathy. To determine how specific these findings are to Alzheimer's disease, other neurologic disorders with neurofibrillary tangles and filamentous inclusions were examined. These included progressive supranuclear palsy, Pick's disease, subacute sclerosing panencephalitis, and postencephalitic parkinsonism. Sulfated GAGs were not demonstrated in the neurofibrillary tangles or filamentous structures in any of these disorders. The relationship of GAGs to the pathology of Alzheimer's disease is discussed as is their possible importance in determining the characteristic morphology of the amyloidotic lesion.
The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.
The cerebrovascular amyloid protein from a case of adult Down's syndrome was isolated and purified. Amino acid sequence analysis showed it to be homologous to that of the beta protein of Alzheimer's disease. This is the first chemical evidence of a relationship between Down's syndrome and Alzheimer's disease. It suggests that Down's syndrome may be a predictable model for Alzheimer's disease. Assuming the beta protein is a human gene product, it also suggests that the genetic defect in Alzheimer's disease is localized on chromosome 21.
A purified protein derived from the twisted beta-pleated sheet fibrils in cerebrovascular amyloidosis associated with Alzheimer's disease has been isolated by Sephadex G-100 column chromatography with 5 M guanidine-HC1 in 1 N acetic acid and by high performance liquid chromatography. Amino acid sequence analysis and a computer search reveals this protein to have no homology with any protein sequenced thus far. This protein may be derived from a unique serum precursor which may provide a diagnostic test for Alzheimer's disease and a means to understand its pathogenesis.
Amyloid in Alzheimer*! disease and unconventional viral infection
- H M Wisniewski
- Iqbauc
- I Grundke-Iqbal
- R Rubeastein
- G Y Wen
- P A Merz
- Kascakjt
- K Kristensson
Wisniewski.H.M., IqbaUC., Grundke-Iqbal.I., Rubeastein.R., Wen,G.Y.,
Merz.P.A., Kascakjt and Kristensson.K. (1986) Amyloid in Alzheimer*!
disease and unconventional viral infection. International Symposium On
Dementia and Amyloid Neuropathohgy, Suppl. 3, 87-94.
Ultrastructural irnmunolocalization of heparan sulfate proteogrycan to amyloid fibrils in the neuritic plaques of Alzheimer's disease
- A D Snow
- H Mar
- D Hocuin
- T N Wight
Snow.A.D., Mar,H., HocUin.D. and Wight,T.N. (1988) Ultrastructural
irnmunolocalization of heparan sulfate proteogrycan to amyloid fibrils in the
neuritic plaques of Alzheimer's disease. J. NeuropathoL Exp. NeuroL, 47,
164A.
Unique distribution of a dennatan sulfaie proteogrycan in the amyloid plaque and neurofibriUary tangles of Alzheimer's disease
- A D Snow
- H Mar
- D Nochlin
- H Kresse
- N Wightj
Snow.A.D., Mar.H., Nochlin.D., Kresse.H. and WightJ.N. (1990b) Unique
distribution of a dennatan sulfaie proteogrycan in the amyloid plaque and
neurofibriUary tangles of Alzheimer's disease. J. Cell Bioi., 111, 267a.