Presence Of Clusterin Research Articles

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Clusterin exacerbates interleukin-1β-induced inflammation via suppressing PI3K/Akt pathway in human fibroblast-like synoviocytes of knee osteoarthritis

This study aimed to examine, a multifaceted chaperon-like protein exerting anti-inflammatory action, clusterin (CLU), mRNA and protein levels in the systemic and local joint environment of knee osteoarthritis (OA) patients and to determine whether CLU inhibited interleukin (IL)-1β-induced inflammation in knee OA fibroblast-like synoviocytes (FLSs) through modulating phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. CLU protein and mRNA expressions in the synovium and its protein levels in plasma and synovial fluid of knee OA patients were measured using immunohistochemistry, real-time PCR, and ELISA, respectively. Anti-inflammatory effect of CLU was further elucidated in knee OA FLSs treated with IL-1β in the absence or presence of CLU, CLU alone, or PI3K inhibitor (LY294002) along with IL-1β and CLU. In a clinical study, compared with knee OA patients without synovitis, CLU protein and mRNA were expressed in the synovium of knee OA patients with synovitis, especially those with high-grade, consistent with analyses of its plasma and synovial fluid levels. CLU mRNA and protein levels were both associated with synovitis severity. An in vitro study uncovered that CLU significantly alleviated IL-1β-induced overproduction of nitric oxide and IL-6 in knee OA FLSs. Furthermore, CLU significantly attenuated inflammation and extracellular matrix degradation induced by IL-1β via down-regulating expressions of IL-6, nuclear factor kappa B, and matrix metalloproteinase-13. Mechanistically, CLU significantly impeded IL-1β-induced Akt phosphorylation in knee OA FLSs, in line with addition of LY294002 along with IL-1β and CLU. These findings suggest that CLU may have potential as a novel therapeutic target for synovitis and cartilage destruction in knee OA.

The circulating molecular chaperone clusterin interacts with amyloidogenic transthyretin oligomers and modulates amyloid formation

Clusterin (CLU) is an abundant, circulating chaperone co‐localizing with amyloid deposits of transthyretin (TTR). The precise interaction of CLU with misfolded, amyloidogenic TTR intermediates has not been defined. In vitro, wild‐type (WT) TTR was unfolded and aggregation induced using NaCl. Cross‐linked aggregation samples from 0–24 h showed a transition from tetramers to oligomers by SDS‐PAGE. Moreover, samples from 0.5 to 4.0 h showed increasing reactivity to the A11 oligomer‐specific antibody. Circular dichroism spectroscopy showed unfolding of native TTR structure, increased beta‐sheet content and aggregate formation. Using surface plasmon resonance (SPR) we investigated the interactions of CLU with surface‐bound native and oligomeric forms of TTR. SPR small molecule binding of Congo red (CR) to oligomers and diflunisal to native tetramers demonstrated the loss of diflunisal binding and increased CR binding to oligomeric TTR. CLU was found to bind to early and late stage oligomers in a concentration dependent manner by SPR. Amyloid formation in the presence of CLU indicated enhanced oligomerization at a 1:1 (TTR:CLU) molar ratio as measured by ThioflavinT binding. Investigation of CLU interactions with oligomeric forms of TTR may provide mechanistic insight for the presence of this chaperone in amyloid deposits. Supported by NIH grant RO1AG031804 (LHC) and the Young Family Amyloid Research Fund.

Increased Production of Clusterin in Biopsies of Repair Tissue following Autologous Chondrocyte Implantation.

Objective. To characterize the immunolocalization of clusterin in the repair cartilage of patients having undergone autologous chondrocyte implantation (ACI) and evaluate correlation to clinical outcome. Design. Full-depth core biopsies of repair tissue were obtained from 38 patients who had undergone ACI at an average of 18 ± 13 months previously (range 8-67 months). The biopsies were snap frozen, cryosectioned, and clusterin production immunolocalized using a specific monoclonal clusterin antibody and compared with normal and osteoarthritic cartilage. Clinical outcome was assessed from patients preoperatively, at the time of biopsy, and annually postoperatively. Results. Intensity of immunostaining for clusterin decreased with age in healthy cartilage tissue. Clusterin was detected to a variable degree in 37 of the 38 ACI cartilage biopsies, in single and clustered chondrocytes, in the pericellular capsule and the cartilage extracellular matrix, as well as the osteocytes and osteoid within the bone. Chondrocytes in hyaline repair tissue were significantly more immunopositive than those in fibrocartilage repair tissue. Clinical outcome improved significantly post-ACI, but did not correlate with the presence of clusterin in the repair tissue. Conclusions. These results demonstrate the presence of clusterin in actively repairing human cartilage and indicate a different distribution of clusterin in this tissue compared to normal cartilage. Variability in clusterin staining in the repair tissue could indicate different states of chondrogenic differentiation. The clinical significance of clusterin within repair tissue is difficult to assess, although the ideal functioning repair tissue morphology should resemble that of healthy adult cartilage.

Presence, localization, and origin of clusterin in normal human spermatozoa

Clusterin in mammalian semen is a secretory form of clusterin (sCLU) with the heterodimeric structure. It is secreted by the epididymis and seminal vesicle. It is generally agreed that clusterin mainly exists on the surface of abnormal spermatozoa and is implicated in decreased sperm motility, sperm aggregation and infertility. However, few studies observe clusterin in normal spermatozoa, which is presumed to be a novel form. Up to now, the systematical information about the presence, localization, origin and function of clusterin in normal human spermatozoa has yet not been established. The aim of our current study is to systematically research clusterin in normal human spermatozoa. We detected the presence of clusterin via western blot, explored the localization of clusterin using immunofluorescence, and investigated the origin and distribution of clusterin in human testis by western blot and immunohistochemistry. We found native clusterin in the inner plasma membrane of normal human spermatozoa. It was derived from the testis and showed similar molecular weight and heterodimeric structure compared with sCLU in semen and on the surface of abnormal spermatozoa. Clusterin in normal spermatozoa should be self-synthesized during the later stage of spermatogenesis. The different localization and origin suggested that the clusterin observed by us may be a novel form compared with conventional sCLU on the surface of abnormal spermatozoa.

Localization of the Potential Zonal Marker Clusterin in Native Cartilage and in Tissue-Engineered Constructs

An increasing number of studies aim to engineer cartilage tissue that more closely mimic the zonal organization of native articular tissue. Further understanding of zonal differences is crucial for successful development and evaluation of such grafts. We therefore aimed to characterize the secretion of the potential zonal marker clusterin by zonal articular chondrocytes in osteoarthritic and healthy articular cartilage and in tissue-engineered constructs. Clusterin secreted by superficial, middle, and deep zones equine chondrocytes was immunolocalized in cytospins of alginate cultured superficial, middle, and deep zones equine chondrocytes. Clusterin was present within the superficial zone of native cartilage; after isolation of the cells from healthy articular cartilage, staining for clusterin was limited to cells derived from the superficial zone. Staining disappeared after expansion, but reappeared during (re)differentiation and was more pronounced within the cultures derived from the superficial zones of the cartilage. The presence of clusterin was associated with clusters of differentiating chondrocytes, rather than highly proliferative cells and did not specifically colocalize with proteoglycan-4. Because staining for clusterin was more abundant in cultures of superficial chondrocytes compared to those of cells of the deeper layers, it may be used to further characterize zonal cartilage constructs.

Clusterin regulates transthyretin amyloidosis

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.

Clusterin expression in cholestasis, hepatocellular carcinoma and liver fibrosis

Clusterin shares functional properties with small heat shock proteins. In contrast to other heat shock proteins, it is present in the extracellular space. Its expression is altered in various diseases. The aim was to evaluate the presence and distribution of clusterin in liver diseases associated with cholestasis, in fibrosis and in hepatocellular carcinoma. Tissue microarrays and biopsy materials were used to evaluate immunohistochemically the expression of clusterin in hepatocellular carcinoma, primary sclerosing cholangitis, primary biliary cirrhosis, mechanical cholestasis, drug-induced cholestasis, liver fibrosis and cirrhosis. The presence of clusterin in human bile was assessed by Western blotting. Furthermore, real-time reverse transcriptase-polymerase chain reaction was performed on liver tissue with mechanical cholestasis. Clusterin colocalized with elastic fibres, but not with collagen, hepatocytes or bile duct epithelia. It was detected in bile plugs in cholestasis and hepatocellular carcinomas with pseudoglandular features within the lumina. Clusterin was demonstrated in bile by Western blotting and its mRNA was expressed in normal and cholestatic livers. Clusterin may protect bile duct epithelium against offensive biliary components or inhibit precipitation of biliary proteins. The association of clusterin with elastic fibres could reflect an extracellular chaperone function by either protecting elastic fibres or shielding abnormal elastic material.

Serum antibodies against prostasomal clusterin in prostate cancer patients

Objective. Clusterin is a ubiquitous secretory sulphated glycoprotein present in prostasomes. It is an anti‐apoptotic mediator in prostate cancer and is among the most frequently occurring prostasomal proteins immunogenic in prostate cancer patients. The aim of the present study was to investigate the occurrence of anti‐clusterin antibodies in the serum of patients with prostate cancer and whether there is a relationship between anti‐clusterin antibody titres and other clinico‐pathological variables. Material and methods. Serum samples were collected from 391 consecutive patients with suspected prostate cancer (150 benign prostate and 241 prostate cancer). The patients' serum samples were used in an ELISA where microtitre wells were coated with purified clusterin from serum of a healthy volunteer. Flow cytometric studies of clusterin and prostasomes were performed. Results. Flow cytometric analyses revealed the presence of clusterin on the surface of seminal prostasomes. Anti‐clusterin ELISA titres in sera of patients did not differ significantly from those of a control group. A significant “inverse” correlation existed between anti‐clusterin ELISA titres and lymph node metastases (p = 0.047), but only 11 out of 161 patients had metastases. These titres correlated significantly with total prostate (p = 0.021) and transitional zone (p = 0.015) volumes of the patients. Conclusions. The correlation between serum anti‐clusterin antibody titres and other clinico‐pathological variables was generally weak in prostate cancer patients, although clusterin has been assigned an important role in tumourigenesis and progression of prostate cancer. However, the anti‐clusterin antibody titre appeared to be related to prostate volume, correlating to both transitional zone volume and total volume of the prostate.

Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos

It has been suggested that spermatozoa can deliver mRNAs to the oocyte during fertilisation. Using reverse transcription and real-time quantitative polymerase chain reaction analysis (RQ-PCR), we evaluated the presence of clusterin (CLU), protamine 2 (PRM2), calmegin (CLGN), cAMP-response element modulator protein (CREM), methyltransferase 1 (DNMT1), linker histone 1 (H1), protamine 1 (PRM1), TATA box-binding protein associated factor 1 (TAF1) and TATA box-binding protein (TBP) in porcine mature oocytes, zygotes and two-cell stage embryos. Spermatozoa isolated from semen samples of boars contained all transcripts investigated, whereas oocytes contained only CREM, H1, TAF1, and TBP mRNAs. The zygote and two-cell stage embryos contained CLU, CREM, H1, PRM1, PRM2, TAF1 and TBP transcripts. Our observations suggest that porcine spermatozoa may delivery CLU, PRM1 and PRM2 mRNAs to the oocyte, which may contribute to zygotic and early embryonic development.

Doxazosin-induced clusterin expression and apoptosis in prostate cancer cells

The purpose of this study was to correlate temporal expression of clusterin and apoptosis in androgen-independent human prostate cancer cells (PC-3) treated with 25 μM doxazosin. DNA fragmentation, reverse transcriptase polymerase chain reaction, and terminal transferase-mediated biotinylated 16-desoxy-uridene triphosphate nick-end labeling (TUNEL) assays were used to assess degree of apoptosis and temporal and spatial expression of clusterin mRNA and protein. DNA fragmentation was significant at 48 hours. Clusterin mRNA expression was 3-fold higher than control at 9 hours and was maintained over 48 hours. The TUNEL assay showed increasing percentage of apoptotic cells and presence of clusterin after doxazosin treatment. During doxazosin-induced apoptosis in PC3 cells, clusterin appeared to initially accumulate in the cytoplasm and protect against apoptosis; later, after its transport to the nucleus, clusterin was no longer able to suppress apoptosis.

Clusterin Associates with Altered Elastic Fibers in Human Photoaged Skin and Prevents Elastin from Ultraviolet-Induced Aggregation in Vitro

Clusterin is a secreted glycoprotein with stress-induced expression in various diseased and aged tissues. It shares basic features with small heat shock proteins because it may stabilize proteins in a folding-competent state. Besides its presence in all human body fluids, clusterin associates with altered extracellular matrix proteins, such as beta-amyloid in Alzheimer senile plaques in the brain. Because dermal connective tissue alterations occur because of aging and UV radiation, we explored the occurrence of clusterin in young, aged, and sun-exposed human skin. Immunohistochemical analysis showed that clusterin is constantly associated with altered elastic fibers in aged human skin. Elastotic material of sun-damaged skin (solar elastosis), in particular, revealed a strong staining for clusterin. Because of the striking co-localization of clusterin with abnormal elastic material, we investigated the interaction of clusterin with elastin in vitro. A chaperone assay was established in which elastin was denatured by UV irradiation in the absence or presence of clusterin. This assay demonstrated that clusterin exerted a chaperone-like activity and effectively inhibited UV-induced aggregation of elastin. The interaction of both proteins was further analyzed by electron microscopy, size exclusion chromatography, and mass spectrometry, in which clusterin was found in a stable complex with elastin after UV exposure.

Clusterin is protective in pancreatitis through anti-apoptotic and anti-inflammatory properties

Clusterin is overexpressed in pancreas during the acute phase of pancreatitis. We intended to clarify the role of clusterin expression in stressed exocrine pancreas. We performed in vitro experiments in transfected AR4-2J cells with modified expression levels of clusterin and in vivo studies in clusterin-deficient mice. AR4-2J cells were exposed to agents mimicking cell-stress during pancreatitis (cerulein, hydrogen peroxide, staurosporine or lysophosphatidylcholine). Clusterin-overexpressing AR4-2J cells showed higher viability after cell stress and accordingly reduced rates of apoptosis and lessened caspase-3 activation. Blockage of endogenous clusterin expression reduced viability and enhanced apoptosis. Presence of clusterin reduced NF-κB activation and expression of the NF-κB target genes TNF-α and MOB-1 under cell stress. Clusterin-deficient mice showed a more severe course of acute experimental pancreatitis with enhanced rates of apoptosis and inflammatory cell infiltration. We concluded that clusterin was protective during inflammation of exocrine pancreas because of its anti-apoptotic and anti-inflammatory functions.

The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins

We have shown previously that a 'soluble' form of PrP (prion protein), not associated with membranous vesicles, exists in the male reproductive fluid [Ecroyd, Sarradin, Dacheux and Gatti (2004) Biol. Reprod. 71, 993-1001]. Attempts to purify this 'soluble' PrP indicated that it behaves like a high-molecular-mass complex of more than 350 kDa and always co-purified with the same set of proteins. The main associated proteins were sequenced by MS and were found to match to clusterin (apolipoprotein J), BPI (bacterial permeability-increasing protein), carboxylesterase-like urinary excreted protein (cauxin), beta-mannosidase and beta-galactosidase. Immunoblotting and enzymatic assay confirmed the presence of clusterin and a cauxin-like protein and showed that a 17 kDa hydrophobic epididymal protein was also associated with this complex. These associated proteins were not separated by a high ionic strength treatment but were by 2-mercaptoethanol, probably due to its action on reducing disulphide bonds that maintain the interaction of components of the complex. Our results suggest that the associated PrP retains its GPI (glycosylphosphatidylinositol) anchor, in contrast with brain-derived PrP, and that it is resistant to cleavage by phosphatidylinositol-specific phospholipase C. Based on these results, the identity of the associated proteins and the overall biochemical properties of this protein ensemble, we suggest that 'soluble' PrP can form protein complexes that are maintained by hydrophobic interactions, in a similar manner to lipoprotein vesicles or micellar complexes.

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of α-lactalbumin, γ-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α-lactalbumin. Reduction-induced changes in the conformation of α-lactalbumin, in the absence and presence of clusterin, were monitored by real-time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and γ-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein.

Injury-induced synthesis and release of apolipoprotein E and clusterin from rat neural cells.

Apolipoproteins in the brain have assumed major clinical importance since it was shown that one of the allelic forms of apolipoprotein E, apoE-4, is a risk factor for Alzheimer's disease. Using tissue culture of embryonic rat spinal cord, we examined the effect of neuronal injury on the up-regulation of two apolipoproteins, apolipoprotein E and clusterin (apoJ). In order to study the influence of neuronal cells, we exploited the specific neurotoxic effect of elevated glutamate on these cells. Overstimulation by excess glutamate induced neuronal degeneration as assessed by morphological and biochemical criteria, notably the activity of choline acetyltransferase, which serves as a marker for cholinergic neurons. High concentrations of glutamate increased mRNA synthesis and the production and secretion of both apolipoprotein E and clusterin protein. Both neuronal cell death and release of the peptides were calcium-dependent and could be blocked by the NMDA receptor antagonist MK-801. Immunohistochemical data revealed the presence of clusterin in both neuronal and non-neuronal cells whereas apolipoprotein E was mainly expressed in non-neuronal cells. The results are suggestive of concerted up-regulation of apolipoprotein E and clusterin when neural cells are subjected to injury.

Immunocytochemical Colocalization of Clusterin in Apoptotic Photoreceptor Cells in Retinal Degeneration SlowrdsMutant Mouse Retinas

In therdsmutant mouse the photoreceptor cells differentiate normally for the first few postnatal days, with the inner segments projecting an extended cilium. However, outer segments fail to form and only rudimentary disks and opsin-laden vesicles assemble at the tip of the cilium. These are shed into the interphotoreceptor space where they are phagocytosed by the retinal pigment epithelial cells. In this animal model, the photoreceptors undergo a slow degeneration via apoptosis leading to eventual loss of the entire photoreceptor population. Since increased expression of clusterin has been implicated in apoptosis, we studied the expression of clusterin in therdsmutant mouse retina and compared it to normal BALB/c retinas. Small intestinal microvillus epithelium was used as a positive control tissue for apoptosis. Immunocytochemistry revealed the presence of clusterin in the ganglion cell, inner nuclear and outer plexiform layers and in the retinal pigment epithelium of both therdsand the BALB/c retinas. Interestingly, scattered clusterin-positive cells were observed in the outer nuclear layer (onl) of dystrophic retinas. Since the increased presence of clusterin protein in the onl of dystrophic retina may indicate dying photoreceptor cells due to apoptosis, we utilized a co-localization procedure for apoptotic nuclei and clusterin. For apoptosis we utilized an in situ 3′ end labeling of fragmented DNA (TUNEL) and immunohistochemistry for clusterin using brown and red colored substrates respectively. Small intestine tissue sections were also included as positive controls for apoptosis. Our results show that clusterin is co-localized with apoptotic nuclei both in the onl ofrdsmutant retinas as well as in the small intestine epithelial cells undergoing cell turnover and exfoliation. These results are of interest since overexpression of clusterin is also observed in other neuro-degenerative diseases such as Alzheimer's and Pick's disease.

Clusterin (apoJ) Alters the Aggregation of Amyloid β-Peptide (Aβ 1-42) and Forms Slowly Sedimenting Aβ Complexes That Cause Oxidative Stress

Clusterin (apoJ), a multifunctional apolipoprotein made by cells in the brain and many other locations, is associated with aggregated amyloid β-peptide (Aβ) in senile and diffuse plaques of Alzheimer's disease (AD). We observed that purified human serum clusterin partially blocked the aggregation of synthetic Aβ 1-42, as shown by centrifugal assays (14,000g × 10 min) and by atomic force (scanning probe) microscopy. Slowly sedimenting Aβ complexes were formed in the presence of clusterin, which included aggregates >200 kDa that resist dissociation by low concentrations of SDS, Clusterin enhanced the oxidative stress caused by Aβ, as assayed by oxidative stress in PC12 cells with MTT, which is widely used to estimate neurotoxicity. These indications of enhanced neurotoxicity by the MTT assay were observed in the highly aggregated rapidly sedimenting fraction, but also in more slowly sedimenting "soluble" forms. This novel activity of slowly sedimenting Aβ may enhance the neurotoxicity of Aβ deposits in AD brains, because soluble complexes have a potential for diffusing to damage distal neurons.

Competition between cell-substratum interactions and cell-cell interactions.

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.