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Amyloid, 2012; 19(S1): 3–4© 2012 Informa UK, Ltd.ISSN 1350-6129 print/ISSN 1744-2818 onlineDOI: 10.3109/13506129.2012.675370

Extracellular chaperones, such as clusterin, have been proposed to contribute to extracellular protein homeostasis. In Alzheimer’s disease (AD), clusterin was suggested to be involved in fibrillogenesis and extracellular misfolded protein clearance. Here, we study the relevance of clusterin in familial amyloidotic polyneuropathy (FAP). Analysis of clusterin levels by ELISA showed decreased levels in asymptomatic and in FAP patients, which could be regarded as a consequence of clusterin mobilization to tissues. Incubation of a neuroblastoma cell line with transthyretin (TTR) oligomers resulted in secretion of clusterin into the media and binding to oligomeric species, not observed in parallel experiments with soluble TTR. Overall, our results allow us to postulate a putative protective role of clusterin in FAP, namely in the modulation of TTR aggregate formation. Future experiments are required to clarify the role of clusterin in FAP.

Keywords: Amyloid, clusterin, heat shock, transthyretin

Introduction

Intracellularly, molecular chaperones are capable to repair or target damaged proteins to degradation; a corresponding extracellular quality control mechanism is thought to exist where extracellular chaperones are major players by bind-ing to extracellular misfolded proteins and promoting their endocytosis for intracellular degradation. This mechanism could be relevant for a disease like familial amyloidotic polyneuropathy (FAP) which is characterized by extracel-lular deposition of amyloidogenic mutant transthyretin (TTR) particularly in the peripheral nervous system (PNS) [1]. Clusterin, a protein present in most physiologic fluids, has been suggested to have an extracellular quality control function [2]. It was described to act as a molecular chaper-one in the extracellular milieu, inhibiting protein aggregation

in an ATP independent manner after binding to misfolded proteins forming soluble, high molecular weight complexes maintaining non-native proteins in a stabilized state until heat shock protein 70 can refold them [3]. Available data in Alzheimer’s disease (AD) support the notion that besides a role as a molecular chaperone, stabilizing proteins and inhib-iting their aggregation, clusterin also has an important role in the clearance of extracellular aggregates by endocytosis. Here, we investigate clusterin relevance in FAP.

Material and methods

Elisa for clusterin of human FAP plasmaHuman clusterin levels were analyzed in plasma of FAP 0 (asymptomatic carriers) FAP patients and normal indi-viduals using the Human Clusterin Competitive ELISA kit (AdipoGen), following the manufacturer instructions.

Cell culture assays: dot-blot assay in nitrocellulose and cellulose acetate membranesSH-SY5Y human neuroblastoma cells were cultured, and incubated for 18 h with oligomeric and soluble TTR (0.110 mg/ml). Cell media was analyzed by dot blot using either nitrocellulose or cellulose acetate membranes.

Results and discussion

Previous studies showed that intracellular molecular chaperones, such as heat shock proteins, are upregulated in FAP most likely through HSF1. Extracellular molecular chaperones have not been fully explored for TTR-related disorders, namely their expression and effect in extracellular TTR aggregation. To study clusterin relevance in FAP, we first analyzed the levels of clusterin in plasma of asymptomatic carriers (FAP 0) and FAP patients. We observed by ELISA that

The heat shock response in FAP: the role of the extracellular chaperone clusterin

Joana Magalhães1,2 & Maria João Saraiva1,2

1IBMC, Instituto de Biologia Molecular e Celular, Porto, Portugal and 2ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal

Correspondence: M. J. Saraiva, Molecular Neurobiology, IBMC R. Campo Alegre 823, 4150, Porto, Portugal. Tel: 351-22-6074900. Fax: 351-22-6099157. E-mail: mjsaraiv@ibmc.up.pt

Amyloid

2012

19

S1

3

4

© 2012 Informa UK, Ltd.

10.3109/13506129.2012.675370

1350-6129

1744-2818

Clusterin in FAP

J. Magalhães & M. J. Saraiva

09March2012

(Accepted March 2012)

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4 J. Magalhães & M. J. Saraiva

Amyloid

clusterin levels decreased in FAP patients (Figure 1) as well as in asymptomatic carriers; decreased clusterin levels could be related to mobilization of clusterin to tissues. Alternatively, it might be related to antigen accessibility, as clusterin could bind modified TTR and affect access of the clusterin antibody.

We have shown, by double immunohistochemistry, that clusterin associates with both fibrillar and nonfibrillar TTR deposits in human nerve and that clusterin is present in TTR fibrils extracted from human kidney [4]. The association of clusterin with TTR deposits at sites of amyloid and nonfibril-lar deposition lead us to hypothesize that clusterin could bind to TTR aggregates for stabilization and inhibition of further aggregation, as clusterin does for amyloid β in AD.

To further explore the putative neuroprotective role of clusterin in FAP, particularly in the modulation of TTR aggregation and cellular effects we performed in vitro stud-ies. First, we observed that incubation of SH-SY5Y cells with TTR oligomers leads to intracellular clusterin overexpression [4]. Moreover, clusterin secretion levels were also analyzed and a significant increase in clusterin in the medium of cells incubated with TTR oligomers was observed (Figure 2A). All together, these results indicate that oligomeric TTR induce overexpression and secretion of clusterin in our cellular model.

We then investigated by cellulose acetate dot-blot filter assay, the association of clusterin to TTR oligomers in media of cells incubated with TTR oligomers and observed staining for clusterin in the retained aggregates (Figure 2B). This result provided further evidence that clusterin binds to extracellu-lar TTR aggregates. Furthermore, clusterin modulates TTR

aggregation, as clusterin deprivation in cellular models stimu-lates extracellular TTR aggregation [4].

Two theories have been proposed in general for clusterin presence in protein deposits: clusterin may be in the deposits either as a consequence of a failed response to prevent aggre-gation or as a part of the aggregation process itself. Our data suggest that in FAP clusterin associates with TTR extracel-lular aggregates and can influence TTR aggregation; however, details of the interaction of clusterin with TTR aggregates and the clusterin role in the clearance of extracellular misfolded TTR needs further clarification.

Acknowledgements

We thank Tânia Ribeiro, Paula Gonçalves, and Paul Moreira from IBMC for animal maintenance, tissue processing and TTR production, respectively.

Declaration of Interest: This study was supported by Fundação para a Ciência e Tecnologia (FCT) through fellowship SFRH/BD/35980/2007, grant PTDC/SAU-ORG/11313/2009 and FEDER funds through the COMPETE program.

References 1. Saraiva MJ, Birken S, Costa PP, Goodman DS. Amyloid fibril protein

in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin). J Clin Invest 1984;74:104–119.

2. Yerbury JJ, Stewart EM, Wyatt AR, Wilson MR. Quality control of pro-tein folding in extracellular space. EMBO Rep 2005;6:1131–1136.

3. Poon S, Easterbrook-Smith SB, Rybchyn MS, Carver JA, Wilson MR. Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state. Biochemistry 2000;39:15953–15960.

4. Magalhães J, Saraiva MJ. Clusterin overexpression and its possible pro-tective role in transthyretin deposition in familial amyloidotic poly-neuropathy. J Neuropathol Exp Neurol 2011;70:1097–1106.

Figure 1. Clusterin levels in plasma of normal individuals (n = 9), FAP 0 (asymptomatic carriers, n = 9) and FAP patients (n = 9). Comparing to control (normal individuals), FAP patients have a significantly decrease of clusterin levels, while FAP 0 show a decreased tendency.

Figure 2. Association of clusterin with TTR oligomers. (A) Human neu-roblastoma media analysis: nitrocellulose dot-blot assay and clusterin immunodetection of SH-SY5Y medium incubated with TTR species show increased clusterin secretion in cells incubated with TTR oligomers (oTTR) as compared to controls (nontreated, NT and soluble TTR, sTTR). *p < 0.05. (B) Cellulose acetate dot-blot filter assay and clusterin immuno-detection of medium of SH-SY5Y incubated with TTR species. Clusterin is retained in the aggregates present in the medium of cells incubated with oTTR for 16 h. Results were confirmed by three independent experiments.

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