Alzheimer disease-associated cystatin C variant undergoes impaired secretion

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Neurobiology of Disease 13 (2003) 15–21

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Alzheimer disease-associated cystatin C variant undergoes impaired secretion Luisa Benussi,a Roberta Ghidoni,a Tiana Steinhoff,b Antonella Alberici,a Aldo Villa,a Federica Mazzoli,a Francesca Nicosia,a Laura Barbiero,a Laura Broglio,a Enrica Feudatari,a Simona Signorini,a Ulrich Finckh,c Roger M. Nitsch,b and Giuliano Binettia,* a Neurobiology Lab, IRCCS Centro San Giovanni di Dio, Italy Department of Psychiatry Research, University of Zurich, Switzerland c Department of Human Genetics, University Hospital, Hamburg-Eppendorf, Germany b

Received 2 August 2002; revised 6 December 2002; accepted 6 January 2003

Abstract CST3 is the coding gene for cystatin C (CysC). CST3 B/B homozygosity is associated with an increased risk of developing Alzheimer disease. We performed CysC analysis on human primary skin fibroblasts obtained from donors carrying A/A, A/B, and B/B CST3. Pulse-chase experiments demonstrated that the release of the B variant of CysC has a different temporal pattern compared to that of the A one. Fibroblasts B/B homozygous displayed a reduced secretion of CysC due to a less efficient cleavage of the signal peptide, as suggested by high-resolution Western blot analysis and by in vitro assay. In the brain, the reduced level of CysC may represent the molecular factor responsible for the increased risk of Alzheimer disease. © 2003 Elsevier Science (USA). All rights reserved.

Introduction In most of the investigated human body fluids cystatin C (CysC) is established to be the predominant cysteine protease inhibitor. It is found at particularly high concentration in the cerebrospinal fluid (CSF) of the central nervous system (Lofberg and Grubb, 1979) and is active on several proteases, including cathepsins B, H, and L (Barrett et al., 1984). In the brain CysC is synthesized by the choroid plexus and leptomeningeal cells and is localized in both glial cells and neurons (Ohe et al., 1996; Yasuhara et al., 1993; Lignelid et al., 1997). Acute brain injuries, including ischemia, axotomy, or surgery, induce an increase of CysC expression levels, mainly in activated glial cells (Palm et al., 1995; Ishimaru et al., 1996; Miyake et al., 1996; Katakai et al., 1997). These observations suggest that CysC might be in* Corresponding author. IRCCS Centro S. Giovanni di Dio, Neurobiology Lab, Alzheimer’s Disease Unit. Via Pilastroni 4, 25123 Brescia, Italy. Fax: ⫹39-030-3533513. E-mail address: [email protected] (G. Binetti).

volved in neuroregeneration or neurodegeneration of neurons in response to cellular damage. In accordance to this model it has been recently demonstrated that CysC has a proliferative effect on neural stem cells in vitro and in vivo (Taupin et al., 2000). This important role of CysC in the control of cell proliferation and survival is supported by the evidence that tumor growth is reduced in the CysC knockout mouse model (Huh et al., 1999). CysC is also associated to chronic neurodegenerative processes. The accumulation of mutated CysC (Leu68Gln) is known to be the cause of cerebral amyloid formation in patients with heritable cerebral hemorrhage with amyloidosis of the Icelandic type angiopathy (HCHWA-I) (Ghiso et al., 1986; Levy et al., 1989). In the brain of patients with Alzheimer disease (AD), neuronal concentration of CysC protein is increased (Yasuhara et al., 1993) and its association to ␤-amyloid has been established (Vinters et al., 1990; Levy et al., 2001). Further studies revealed that the elevation of CysC in AD is specifically associated to neurons more susceptible to neurodegeneration. As already demonstrated for acute injury models, the prominent cellular source of CysC are glial

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cells (Deng et al., 2001). In accordance with these reports, high levels of CysC were also detected in activated astrocytes throughout the brain of the transgenic mice expressing the Swedish APP mutation (Steinhoff et al., 2001). The involvement of CysC in the pathogenesis of AD is further illustrated in two studies in which the KspI restriction site polymorphism in CTS3, the gene coding for CysC, was associated with an increased risk of developing lateonset AD (Crawford et al., 2000; Finckh et al., 2000). CST3 is present within the population in two common haplotypes, named A and B in Finckh, differing in three strongly genetically linked base substitution in the 5⬘ region of the gene, one laying in the coding region at position 1 of codon 25 (Ala25). The allelic haplotype B, containing nucleotide A at this position (Thr25), was proposed to be a recessive risk allele for late-onset AD (Finckh et al., 2000). Further case-control studies in different populations controversially confirmed or not this genetic association to AD (Roks et al., 2001; Maruyama et al., 2001; Beyer et al., 2001; Dodel et al., 2002). The aim of our study is to address whether the different CST3 haplotypes have any influence on CysC protein, to identify possible biological mechanisms that might explain the association between the presence of the B haplotype and the risk to develop AD pathology.

Methods CST3 genotyping and sequencing DNA from fibroblasts were analyzed for CST3 haplotypes at the 5⬘ end as described by Finckh. All exons of the CST3 gene were amplified with primers derived from 5⬘ and 3⬘ intronic sequence using the amplification program used for CST3 genotyping (Finckh et al., 2000). Amplification conditions were as follows: 50 ng of genomic DNA were amplified in a final volume of 50 ␮l containing 20 pmoles of each primer, 200 ␮M of each dNTP (Invitrogen Corporation, San Diego, CA), 10 mM Tris-HCl (Sigma-Aldrich Corporation, St. Louis, MO), pH 8.3, 50 mM KCl (Sigma-Aldrich Corporation, St. Louis, MO), 1.5 mM MgCl2 (Sigma-Aldrich Corporation, St. Louis, MO), 10% dimethyl sulfoxide (DMSO, SigmaAldrich Corporation, St. Louis, MO) 1.5 units AmpliTaq Gold DNA Polymerase, Applera Corporation-Applied Biosystems, Foster City, CA, (Perkin Elmer). Polymerase chain reaction (PCR) reactions were analyzed on 2% agarose gel to verify the size of the amplified sequence. PCR products were purified on multiscreen-PCR 96-well plate from Millipore Billerica, MA. Reaction sequences were performed using the sequence reaction kit by following manufacturer instructions. Unincorporated big-dye terminators were removed from products by sequencing reaction clean-up multiscreen 96-well filtration plates (Millipore). Purified sequencing reactions were run onto automated DNA sequencer ABI Prism Genetic Analyzer 310

and analyzed on Sequence Navigator Software (Applera Corporation-Applied Biosystem, Foster City, CA). Human primary skin fibroblasts culture and cell treatment Skin fibroblasts primary cultures were selected from the tissue repository of our Institute on the bases of their CST3 genotypes: we cultured cells derived from 11 donors, four being CST3 B/B, four A/B, and three CST3 A/A. Two of B/B subjects were male, all the other donors were female. All subjects were comparable for demographic variables (mean age: 77.75 ⫾ 8.69, 76.25 ⫾ 7.45, and 63 ⫾ 10.82 years; P ⬎ 0.1; education: 5 ⫾ 0, 5.5 ⫾ 0.71, and 5 ⫾ 2.8 years; P ⬎ 0.5 for A/A, A/B, and B/B, respectively). Establishment of fibroblast cultures, growth, and storage conditions are described elsewhere (Benussi et al., 1998). The different fibroblast cell cultures, each at the same passage number, were grown to complete confluence and incubated in serum-free medium for 14 –16 h. Pulse-chase experiments B/B and A/A cell cultures were metabolically radiolabeled for 20 min with the addition of [35S]methionine/cysteine to the medium followed by chase times of various length, as previously described (Wei et al., 1998). CysC was recovered from cell lysates and conditioned media by immunoprecipitation with anti-cystatin C antiserum and protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to x-ray films. The bands were quantified using the NIH Image program: means of densitometric measurements of three experiments were compared by t test for independent samples. Protein extracts preparation and Western blot analysis Conditioned media were collected, cleared from cells debris at 3,000 ⫻ g for 10 min, lyophilized then concentrated by dry-vacuum to be run on Western blot. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and collected in lysis buffer [100 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1% (vol/vol) Igepal CA-630] supplemented with protease inhibitors. Cells were homogenized using 1 ml syringe, then centrifuged at 60,000 ⫻ g for 20 min at ⫹4°C. Protein concentrations were determined by using the BCA assay (Pierce Biotechnology, Rockford, IL). Conditioned media, normalized with respect to protein concentration, and twenty micrograms of cellular lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then subjected to Western blot analysis, as already described (Benussi et al., 1998) using Up-State CysC polyclonal antiserum at 1:1000 dilution. For higher resolution of cellular CysC precast 16% Tris-Tricine gels (Novex Invitrogen Corporation, San Diego, CA) were used. Densitometric analyses were performed by using NIH Image software; means of densitometric measurements normalized by the maximal value were compared by t test for independent samples.

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Plasmids construction and in vitro assay CysC A cDNA was kindly provided by Magnus Abrahamson; CysC B was created by site-directed mutagenesis. The respective signal peptide coding fragments corresponding to allelic variants A (signal peptide A: SPA) and B (signal peptide B: SPB) were amplified by PCR using the following primers: fwd: 5⬘-CGTCCTAGCCGACCATGG-3⬘, rev: 5⬘-CGTCCATGGGGCCTCCCAC-3⬘, and cloned in frame at the 5⬘end of the cDNA coding for green fluorescent protein (CTF-GFP fusion, Invitrogen Corporation, San Diego, CA). The constructs were checked by sequence and then they were expressed by using TNT Rabbit Reticulocyte Lysate system (Promega, Madison, WI) in the presence or in the absence of canine pancreatic microsomal membranes (MM) (Promega). To compare cleavage of SPA and SPB we optimized translation and coprocessing conditions: reactions were performed at 30°C, for 60 min, in the presence of 1 ␮l MM. In vitro reaction products were separated onto a 15% Tris-glycine gel electrophoresis, blotted, and analyzed by using the polyclonal rabbit GFP antiserum at 1:5000 dilution (Invitrogen Corporation, San Diego, CA). Ratio of the densitometric measurements of UC and C bands were compared by t test for independent samples.

Results Pulse-chase experiments show a different kinetic between cystatin C A and B variants Primary skin fibroblasts were cultured to determine whether the different CST3 genotypes might be associated with differences in CST3 gene expression or CysC metabolism. Northern blot analysis of CST3 expression revealed no significant difference in CysC mRNA levels depending on different CST3 genotypes (data not shown). To examine the temporal profile of CysC production and secretion, pulse-chase studies were carried out in A/A and B/B fibroblasts (Fig. 1). Cells were metabolically radiolabeled for 20 min with the addition of [35S]methionine/cysteine to the medium as previously described (Wei et al., 1998), followed by chase for various time lengths (0, 30, 60, 90, 120, 180, 240, and 300 min). Pulse-chase analysis of cellular lysates revealed that almost 100% of CysC intracellular levels are retained in B/B fibroblasts for about 2 h into the chase, while the amount of CysC in A/A cells already decreases to 87% in this elapse of time (Fig. 1B); after 120 min, the amount of CysC was significantly higher in B/B compared to A/A, i.e., 5.06 ⫾ 0.13 and 3.87 ⫾ 0.43, respectively (n ⫽ 3, P ⫽ 0.01). At all the other time points, CysC values were always higher in B/B compared to A/A, but they did not reach a statistically significant difference. In the extracellular compartment, we detected a different kinetic in CysC accumulation, i.e., levels of CysC measured after 60, 90, and 120 min into chase were significantly lower in B/B conditioned media compared to the A/A ones (n ⫽ 3; P ⫽

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0.02, P ⫽ 0.007, P ⫽ 0.034, respectively) (Fig. 1C). CysC concentrations were lower in B/B also after 180 and 240 min into the chase, even if the differences were not statistically significant; after 300 min a difference in CysC levels achieved again a statistical significance (n ⫽ 3, P ⫽ 0.048). Detection of a reduced CysC secretion in human primary skin fibroblasts, specifically associated to B haplotype Western blot analysis from media conditioned for 14 –16 h revealed that the group of the CST3 B/B fibroblasts secreted significantly less CysC into the culture medium compared to the CST3 A/A (Fig. 2, lanes 8 –11 and 1–3, respectively): A/A 0.76 ⫾ 0.22, n ⫽ 18; B/B 0.35 ⫾ 0.25, n ⫽ 20; P ⬍ 0.001, t test. CysC secretion from heterozygous A/B fibroblasts was also analyzed: two of four cultures secreted high amounts of CysC, and the other two secreted low amounts (Fig. 2, lanes 4 –7). CysC levels in the lysates were not significantly different in B/B fibroblasts compared to A/A group: A/A 0.61 ⫾ 0.30, n ⫽ 15; B/B 0.66 ⫾ 0.30, n ⫽ 17; P 0.646, t test. Sequencing of the CST3 coding regions was performed to exclude the presence of other polymorphic sites, which could participate to the alteration in the CysC processing. Exons 1, 2, and 3 were amplified and sequenced; however, no additional polymorphisms were found. Reduced cleavage of the CysC variant B signal peptide A defective processing of CysC variant B was suggested by high-resolution Western blot analysis of B/B fibroblasts lysates, which showed, in addition to normally processed protein, a CysC-immunoreactive band migrating at slightly higher molecular weight (Pre-CysC; Fig. 3). CysC A and B variant processing was tested in an in vitro assay: The SP cDNA sequence corresponding to allelic variants A and B was subcloned in frame with the cDNA of GFP (Invitrogen Corporation, San Diego, CA), generating the respective constructs SPA-GFP and SPB-GFP. The in vitro translation of the constructs revealed in both cases a protein of ⬃28 kDa (Un-Cleaved, UC, in Fig. 4, lanes 1 and 3); whereas the translation performed in the presence of microsomal membranes (MM) due to the chimera protein undergoing the posttranslational signal peptide cleavage process resulted in a band of ⬃24 kDa (Cleaved, C, on Fig. 4, lanes 2 and 4). Cleavage of CysC signal peptide was less efficient in the SPB-GFP construct compared to the SPA-GFP. Densitometric analysis revealed a cleavage rate of 64.47 ⫾ 6.12% of SPA and of 29.01 ⫾ 7.18% of SPB (n ⫽ 4 P ⬍ 0.001).

Discussion Allelic variants of CST3 polymorphism A and B predicting the Ala25Thr substitution in the signal peptide (SP) of CysC were shown to be associated with an increased risk for

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Fig. 1. Pulse-chase profile of CysC in A/A and B/B fibroblasts. (A) Cells were metabolically radiolabeled for 20 min with the addition of [35S]methionine/cysteine to the medium followed by chase for various length times as detailed at the top of the figure. (B and C) The bands were quantified by using NIH Image program. The mean of the band intensity (n ⫽ 3) for cell lysates (B) and for conditioned media (C) are shown: CST3 A/A fibroblasts (●); CST3 B/B fibroblasts (Œ). t test comparison revealed a statistically significant difference between CST3 A/A and B/B fibroblasts in labelled CysC recovered from the lysates after 120 min into chase (A/A 3.87 ⫾ 0.43, B/B 5.06 ⫾ 0.13) and from the medium after 60, 90, 120, and 300 min into the chase (A/A 5.28 ⫾ 0.80, B/B 3.19 ⫾ 0.55; A/A 5.72 ⫾ 0.27, B/B 3.91 ⫾ 0.18, A/A 6.03 ⫾ 0.32, B/B 4.08 ⫾ 0.59; A/A 6.16 ⫾ 0.13, B/B 4.38 ⫾ 1.09, respectively). *P ⱕ 0.05; **P ⱕ 0.01.

late-onset sporadic AD (Crawford et al., 2000; Finckh et al., 2000; Beyer et al., 2001). The aim of this study was to determine whether CST3 variants are associated with differences in CysC protein expression or metabolism.

To achieve this goal we cultured primary skin fibroblasts from genotyped subjects carrying either the CST3 A/A, A/B, or B/B haplotypes. Primary fibroblasts were chosen for two reasons. First, as the genetic effect of B variant is

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Fig. 2. CysC secretion by CST3 A/A, A/B, and B/B human primary fibroblasts cultures. CysC level was analyzed in media conditioned by human primary fibroblasts, obtained from 3 carrying CST3 A/A (lanes 1–3), 4 carrying CST3 A/B (lanes 4 –7), and 4 being CST3 B/B donors (lanes 8 –11). The different fibroblast cell cultures, each at the same passage number, were grown to complete confluence and incubated in serum free medium for 14 –16 h. Conditioned media were cleared from cells debris at 3000 ⫻ g for 10 min, concentrated by dry-vacuum, and separated by 16% Tris-glycine SDS-PAGE. Samples were normalized with respect to cellular lysate concentration. Western blot analysis by using polyclonal anti-human CysC antiserum (UpState) revealed an immunoreactive band migrating at around 14 kDa. CysC levels are highly reduced in B/B fibroblasts (lanes 8 –11), compared to A/A cells (lanes 1–3). Two of 4 heterozygous A/B fibroblast cultures secreted high amounts of CysC (lanes 5 and 6), and two secreted low amounts (lanes 4 and 7). Human embryonic kidney (HEK) conditioned medium was loaded as a positive control (lane 12).

recessive, we thought that by using a transfection approach its phenotype might be masked by the presence of the endogenous variant; second, high levels of expression of CysC in cells permanently transfected result in its aggregation (Wei et al., 1998; Merz et al., 1997); this biochemical modification could also hide the phenotypic effect of the B variant. Since no difference in messenger RNA levels was associated to the polymorphisms in the 5⬘ end of the CST3 gene, we investigated if the polymorphism in the coding region of CST3, predicting an Ala-to-Thr substitution, could modify indeed the protein metabolism. We analyzed by pulse-chase

Fig. 3. High-resolution Western blot analysis of intracellular CysC. Twenty micrograms of total lysate from fibroblasts were run in a 16% Tris-Tricine precast gel (Invitrogen Corporation, San Diego, CA). Western blot analysis using CysC antiserum reveals in B/B (lanes 1–3) a band migrating higher than the most prevalent band (Pre-CysC) that is solely present in the A/A lysates (lanes 4 and 5). No CysC aggregates were detected even after boiling the samples.

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Fig. 4. In vitro analysis of the CysC signal peptide processing, variants A and B. SP-GFP was translated in vitro, using Promega’s TNT Rabbit Reticulocyte Lysate system, for 60 min in the absence (lanes 1 and 3) or in the presence of 1 ␮l of Microsomal Membranes (MM) (lanes 2 and 4). In vitro product was run in a 15% self-made Tris-glycine gel, and then analyzed by Western blot, by using green fluoroscent protein (GFP) antiserum from Invitrogen Corporation, San Diego, CA. Translated protein migrates at ⬃28 kDa (UC ⫽ uncleaved); processing by micromosomal membranes (MM) generates a band of ⬃24 kDa (C ⫽ cleaved). Cleavage of SPB-GFP (lane 4) was significantly less efficient than the one of SPA-GFP (lane 2); SPA cleavage: 64.47 ⫾ 6.12%; SPB: 29.01 ⫾ 7.18%; n ⫽ 4, P ⬍ 0.001.

the temporal profile of CysC metabolism in A/A and B/B fibroblasts (Fig. 1). These experiments suggested that the B variant of CysC is retained longer in the cells, whereas the A one is rapidly released into the medium. A significant difference in the extracellular level of CysC was evident within the first 120 min of the experiment; following that time, the kinetic of CysC release was similar, with CysC levels being always lower in B/B compared to A/A. After longer incubation, 300 min into the chase, the level of CysC in B/B medium was once again significantly different from A/A. In view of these data, we can suppose that, in addition to a defect in the secretion, in the medium the B variant of CysC may undergo degradation more rapidly than the A one. Long-term accumulation of CysC was then detected: CysC extracellular level in medium conditioned by B/B cells was highly reduced compared to A/A fibroblasts (Fig. 2). To test whether B/B phenotype was visible even in the presence of a single B allele, CysC was analyzed in medium conditioned by heterozygous cells. Interestingly, high levels of CysC were detected in two cultures, while in the other two CysC secretion was comparable to the one measured in B/B cells. The presence of either an “A” or a “B” phenotype in heterozygous cells suggests that just one of the two CST3 alleles might be alternatively expressed. The mechanism of this control needs to be further investigated, as no effect was attributable to gender or age. CST3 exons sequence analyses of fibroblasts donors confirmed the absence of any other polymorphic sites, which could be associated with reduced CysC secretion. Even if the effect of other polymorphic sites in genes located in the proximity of CST3 cannot be excluded, the following observations strongly suggest that reduced levels of CysC are specifically associated with the B haplotype. The effect on the CysC secretion was attributed to the substitution of the penultimate amino acid of the signal

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peptide. This amino acid exchange was indeed demonstrated to be responsible for a defective maturation of the B isoform, leading to an impaired secretion of the protein. High-resolution Western blot analysis of fibroblasts intracellular lysate revealed in B/B cells a pool of CysC migrating at slightly increased molecular size, suggesting the presence of a CysC portion that retains unprocessed signal peptide. An impaired signal peptide cleavage in the B variant was further demonstrated in an in vitro approach. The signal peptide coding sequence, A and B variant, was subcloned in frame with the GFP protein, generating SPA-GFP and SPBGFP, respectively. Analysis of the processing by microsomal membranes (MM) of the chimera protein revealed that the cleavage of CysC signal peptide was less efficient in the SPB-GFP construct. Taken together these results confirm our hypothesis that an impaired CysC secretion could be attributed to a defective maturation of CysC, due to the amino acid substitution in the signal peptide. This variation, which changes the nonpolar amino acid alanine to a charged threonine, significantly alters the signal sequence hydrophobicity profile, which is known to be more important in the signal-recognition process than the exact amino acid sequence (Alberts et al., 1994). The intracellular cleavage of CysC variant B, which is defective at the predicted signal peptidase site, may occur at an unusual amino acid sequence; this event may cause a secretion of an alternatively processed protein. This protein could be more susceptible to degradation, as suggested by pulse-chase experiments: highly reduced levels of CysC in B/B medium may result from a combination of a defect in the CysC release from the cells and an increased degradation of the secreted protein. The CysC variant with Leu68Gln substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposits in patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I) (Ghiso et al., 1986; Levy et al., 1989). The presence of the Leu68Gln substitution in CysC is associated with its decreased concentration in CSF and leads to its amyloid deposition in the brain (Grubb et al., 1984). Reduced levels of CysC are also present at the peripheral level of Leu68Gln carrying patients: cultured monocytes/macrophages derived from these patients have similar intracellular levels but lower average quantity of CysC in the culture media (Thorsteinsson et al., 1992). Reduced CysC levels both in cell culture and in the CSF of Leu68Gln carrying subjects were attributed to an increased degradation of the mutated protein in the extracellular compartment (Wei et al., 1998). Similarly to what was observed for Leu68Gln variant, reduced CysC levels, detected in our fibroblasts derived from CST3 B/B carrying subjects, might be reflected in an altered metabolism of CysC in the central nervous system. In the brain, the association of the CST3 BB genotype to AD might be due to the defective intracellular processing of CysC. Similarly to what observed in HCHWA-I patients, the production of an altered protein might lead to its self-

aggregation and amyloid formation, which may have a toxic effect on neurons. However, since no CysC intracellular aggregates were detected in our cellular model no experimental evidences support this hypothesis. Alternatively, we can hypothesize that the molecular correlate of the genetic risk conferred by CysC B variant could be associated to the reduction in CysC extracellular levels. The physiological high concentration of CysC in the CSF and its production by choroid plexus, leptomenigeal and glial cells strongly suggest that CysC could exert a protective function in the brain (Lofberg and Grubb, 1979; Ohe et al., 1996; Cole et al., 1989). Several observations support a role of CysC in cell survival: CysC has a proliferative effect on stem cells, in vivo and in vitro (Taupin et al., 2000, Palmer et al., 2001); CysC depletion results in a reduction of tumor cell growth and proliferation (Huh et al., 1999). Following acute injuries, such as ischemia, axotomy, or surgery, CysC levels are increased (Palm et al., 1995; Ishimaru et al., 1996; Miyake et al., 1996; Katakai et al., 1997). Neuronal concentration of CysC protein is increased in activated glia cells in the brain of AD patients (Yasuhara et al., 1993; Deng et al., 2001) and throughout the brain of the Swedish APP mutation transgenic mice (Steinhoff et al., 2001). In AD brain CysC protein is accumulated within intracellular vesicles in the most susceptible neurons (Deng et al., 2001). In view of these experimental evidences, suggesting that CysC could exert a protective role on neurons, we may speculate that the impaired production of CysC in CST3 B/B carrying subjects may predispose them to be more susceptible to neurodegeneration. In addition, CysC is an essential cofactor of FGF2 for the proliferation of rat brain-derived stem cells (Taupin et al., 2000); CysC may be involved in the proliferation of adult neuronal stem cells in the human brain, as already demonstrated for rat CysC on cells derived from postmortem human brains (Palmer et al., 2001). The impaired secretion of CysC observed in CST3 B/B subjects may result in a defective proliferation of stem cells in the brain. Since AD is characterized by continuous loss of neurons not replaced, a failure in neural stem cells replacement may contribute to progression and pathogenesis of this disease. In conclusion, the experimental evidences arising from our data suggest that CST3 B/B might be associated with a reduction in the level of CysC. In the brain, a decreased CysC level may result both in a lack of protection following toxic insults or in a defect in regeneration mediated by stem cells. Both mechanisms could explain how the B variant of CTS3 gene contributes, as a risk factor, to neurodegeneration in sporadic AD.

Acknowledgments Telethon is gratefully acknowledged (Grant E1084). We thank Deborah Simon for careful reading and editing of the manuscript.

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