Overexpression of human cystatin C in transgenic mice does not affect levels of endogenous brain amyloid β peptide

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  • Overexpression of Human Cystatin Cin Transgenic Mice Does Not Affect Levels

    of Endogenous Brain Amyloid PeptideMonika Pawlik,1,4 Magdalena Sastre,1,6 Miguel Calero,5 Paul M. Mathews,2,4

    Stephen D. Schmidt,4 Ralph A. Nixon,24 and Efrat Levy*1,2,4

    Departments of 1Pharmacology, 2Psychiatry, and 3Cell Biology, New York University Schoolof Medicine, New York, NY, 10016; 4Nathan Kline Institute Orangeburg, NY 10962; and

    5Centro Nacional de Microbiologa, Instituto de Salud Carlos III, Madrid, Spain

    Received August 4, 2003; Accepted August 9, 2003

    Abstract

    Cystatin C, an inhibitor of cysteine proteases, colocalizes with amyloid (A) in parenchymal and vascularamyloid deposits in brains of Alzheimers disease (AD) patients, suggesting that cystatin C has a role in AD.Cystatin C also colocalizes with amyloid precursor protein (APP) in transfected cultured cells. In vitro analy-sis of the association between the two proteins revealed that binding of cystatin C to full-length APP does notaffect the level of A secretion. Here we studied the effect of in vivo overexpression of cystatin C on the levelsof endogenous brain A. We have generated lines of transgenic mice expressing either wild-type human cys-tatin C or the Leu68Gln variant that forms amyloid deposits in the cerebral vessels of Icelandic patients withhereditary cerebral hemorrhage, under control sequences of the human cystatin C gene. Western blot analysisof brain homogenates was used to select lines of mice expressing various levels of the transgene. Analysis ofA40 and A42 concentrations in the brain showed no difference between transgenic mice and their nontrans-genic littermates. Thus, in vivo overexpression of human cystatin C does not affect A levels in mice that donot deposit A.

    Index Entries: Alzheimers disease; cystatin C; protease inhibitor; amyloid ; amyloid precursor protein.

    Journal of Molecular NeuroscienceCopyright 2004 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0895-8696/04/22:1318/$25.00

    Journal of Molecular Neuroscience 13 Volume 22, 2004

    Introduction

    Cystatin C (Bobek and Levine, 1992), also knownas trace (Hochwald et al., 1967), is found in all mam-malian body fluids and tissues (Bobek and Levine,1992). In vitro experiments have indicated that it isa cysteine protease inhibitor that inhibits the activ-ities of papain, ficin, and the human cathepsins B,H, and L(Barrett et al., 1984; Bobek and Levine, 1992).Its function in the brain is not well understood, butit has been suggested that cystatin C may have an

    important role in neuronal survival (Palm et al.,1995). A variant cystatin C is the major constituentof amyloid deposited in the brains of patients withhereditary cerebral hemorrhage with amyloidosis,Icelandic type (HCHWA-I) (Gudmundsson et al.,1972), also called hereditary cystatin C amyloidangiopathy (HCCAA) (Olafsson et al., 1996). Amy-loid deposition is restricted to cerebral and spinalarteries and arterioles and leads to recurrent hem-orrhagic strokes early in life (Gudmundsson et al.,1972). The variant cystatin C gene of HCHWA-I

    *Author to whom all correspondence and reprint requests should be addressed. E-mail: elevy@nki.rfmh.org 6Present address: Department of Neurology, University of Bonn, Bonn, Germany.

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    patients has a mutation resulting in a Leu68Gln sub-stitution (Cohen et al., 1983; Ghiso et al., 1986; Levyet al., 1989), which leads to structural differences asso-ciated with increased susceptibility to unfolding,proteolysis, and fibrillogenesis (Calero et al., 2001).

    Amyloid (A) is the major constituent of theamyloid fibrils deposited in the brain of patientswith Alzheimers disease (AD), and in aged indi-viduals without neurologic disorder (Glenner andWong, 1984; Masters et al., 1985). A is a processingproduct of the larger amyloid precursor protein(APP) (Kang et al., 1987). Cleavage by -secretaseresults in the generation of amino-terminal secretedAPP and, together with -secretase, the genera-tion of A. Alternatively, processing by -secretaseprecludes the release of intact A and producessecreted APP (Esch et al., 1990; Sisodia et al., 1990).A has been reported to have heterogeneous car-boxyl termini, with A140 and A142 being the majorspecies produced (Haass et al., 1992; Seubert et al.,1992; Shoji et al., 1992).

    Immunohistochemical studies have revealed thecolocalization of cystatin C with amyloid deposits(Maruyama et al., 1990; Vinters et al., 1990; Itoh etal., 1993; Haan et al., 1994; Levy et al., 2001). Fur-thermore, cystatin C and APP significantly colo-calize in transfected cells (Vattemi et al., 2003) andwithin pyramidal neurons in the cortex of aged indi-viduals and AD patients (Levy et al., 2001). We haverecently shown that cystatin C directly associateswith APP through the extracellular region of A.Nevertheless, in vitro coexpression of either wild-type or variant cystatin C and APP in neuroblas-toma cells did not change A production. Here weprovide in vivo confirmation of the finding that theassociation of cystatin C with APP does not affectA generation. We describe the establishment of cys-tatin C transgenic mouse lines and demonstrate thatoverexpression of human cystatin C in these micedoes not affect the levels of endogenous A40 andA42 in the brain.

    Materials and MethodsGeneration of Cystatin C Transgenic Mice

    Transgenic mice were generated using eitherhuman wild-type or the Leu68Gln variant cystatinC genes (CysC-W and CysC-V, respectively). Vectorsequences were removed by digestion with HindIII.The 8.9-kb full-length human cystatin C genes (Levyet al., 1989) were injected into donor outbred Swiss-Webster single cell embryos in the Skirball Trans-

    genic Facility at New York University School of Med-icine. Swiss-Webster carriers of the transgene werecrossed with C57BL/6 wild-type mice.

    Polymerase Chain Reaction (PCR) Analysis of Tail DNATransgenic mice were identified by amplification

    of a 126-bp DNAfragment unique to the human cys-tatin C sequence from DNAisolated from tails, usingforward 5-ATGGACGCCAGCGTGGAGGA-3 andreverse 5-CTGCTTGCGGGCGCGCAC-3 primers.

    Western Blot Analysis of Mouse Brain HomogenatesMouse brains were homogenized in 150 mM NaCl,

    1% Nonidet P-40, 1% sodium deoxycholate, 0.1%SDS, 10 mM sodium phosphate (pH 7.2), 10 M leu-peptin, 10 M aprotinin, and 2 mM phenylmethyl-sulfonyl fluoride (PMSF). The homogenates werecentrifuged at 10,000g for 15 min, and the super-natant used. Identical amounts of total brain proteinwere applied to each lane of 10% SDS-polyacrylamidegel, confirmed by Western blot analysis with anti--tubulin antibody (1:600; BioGenex Laboratories)and Ponceau Red staining of the membranes. Apoly-clonal anti-cystatin C antibody (1:600; Axell) wasused to identify cystatin C transgene expression.

    Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of AFrozen mouse brains were homogenized in

    sucrose buffer (250 mM sucrose, 20 mM Tris (pH 7.4),1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 M leu-peptin, 10 M antipain HCl, and 10 M pepstatinA), followed by treatment with 0.4% diethylamine,100 mM NaCl, centrifugation at 135,000g, and neu-tralization with Tris-HCl at pH 6.8. Levels of endoge-nous mouse brain A were determined by sandwichELISA as described previously using monoclonalantibodies against the carboxyl terminus of A40(JRF/cA40/10) or A42 (JRF/cA42/26), andhuman A115 (JRF/rA1-15/2) (provided by Dr.Marc Mercken) (Rozmahel et al., 2002).

    Results and DiscussionGeneration of Cystatin C Transgenic Mice

    We have generated transgenic mice expressingeither wild-type or the Leu68Gln variant cystatin Cgenes (CysC-W and CysC-V, respectively). The full-length human cystatin C gene, within an 8.9-kbHindIII fragment, was utilized. It contains the three

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    Journal of Molecular Neuroscience Volume 22, 2004

    exons of the gene, the two introns, and the 5- and3-untranslated regions (Levy et al., 1989). The con-structs were expressed systemically under controlsequences of the human gene. Transgenic mice havebeen identified by amplification of a DNA fragmentof 126 bp unique to the human sequence fromgenomic DNA isolated from tails. The primers useddo not yield a PCR product from DNA of non-transgenic mice (Fig. 1). All founders transmittedthe transgene.

    Selection of Cystatin C Transgenic Mouse LinesWestern blot analysis of brain homogenates was

    used to determine the level of transgene-derived cys-tatin C protein expression. Figure 2 represents a typ-ical Western blot, using an anti-cystatin C antibody.Lines of mice expressing various levels of humanwild-type or variant cystatin C in the brain under-went further study. Swiss-Webster carriers of thetransgene were crossed with C57BL/6 wild-typemice. The fifth generation of crossed mice was testedfor cystatin C expression in the brain. Western blotanalysis of brain homogenates revealed that all ofthe lines preserved the levels of cystatin C overex-pression observed in the founders.

    Western blot analysis of mouse brain homogenatesshowed that mouse cystatin C migrated at about14 kDa (Fig. 2). A minor band at about 20 kDa wasalso observed. As an N-glycosylation consensussequence is present in mouse cystatin C, this bandmay represent glycosylated cystatin C similar to ratcystatin C (Esnard et al., 1990). Rat cystatin C is a13- to 14 kDa basic protein (Turk and Bode, 1991)containing unique consensus sites for N- andO-glycosylation. The existence of a glycosylated formof cystatin C has been reported in rat seminal vesi-cles (Esnard et al., 1988), and this glycosylated formhas been purified from conditioned medium of ratneural stem cell cultures (Taupin et al., 2000). Over-expression of human cystatin C does not correlatewith an increase in the 20-kDa band (Fig. 2), indi-

    cating that the glycosylated band originates frommouse cystatin C. Furthermore, human cystatin Cdoes not have an N-glycosylation consensussequence, suggesting that human cystatin C is notglycosylated in these transgenic mouse lines.

    Characterization of Cystatin C Transgenic MiceStudies of cystatin C transgenic mice were under-

    taken to elucidate the role of increased expressionof this protease inhibitor in vivo and in a variety ofhuman disorders. In addition to the wild-type humangene, we utilized the cystatin C gene containing themutation found in HCHWA-I patients to create atransgenic model of cerebral amyloid angiopathy.We hypothesized that the single amino acid substi-tution in variant cystatin C changes the biology ofthe protein, leading to amyloid fibril formation andearly deposition in brain vessel walls.

    Similar to C57BL/6J homozygous for a null alleleof the cystatin C gene (Huh et al., 1999), the cystatinC transgenic mice are fertile and their appearance isindistinguishable from their nontransgenic littermates.They showed no gross pathological or histopatho-logical abnormalities up to 6 mo of age. Although cys-tatin C null mice are reported to be slightly hypoactive(Huh et al., 1999), no obvious behavioral differenceswere observed in cystatin C transgenic mice comparedto nontransgenic littermates.

    We have set up a colony of aging mice, includingcystatin C transgenic mice and their nontransgenicsiblings. The neuropathological and biochemicalexaminations of these transgenic mice will enablethe in vivo analysis of cystatin C amyloidogenesisand its role in stroke.

    Analysis of Endogenous Brain A LevelsThe levels of endogenous murine A40 and A42

    in brain homogenates were determined by ELISA.Brain homogenates of transgenic mice belonging tothe CysC-V (M11) mouse line were analyzed to obtain

    Fig. 1. Identification of founders by PCR analysis. Ampli-fication products of potential founders of CysC-V transgeniclines (lanes 111). Minus () and plus (+) represent nega-tive and positive controls, respectively.

    Fig. 2. Western blot analysis of cystatin C in brainhomogenates of 3-mo-old offspring of five founders ofCysC-V transgenic lines. Molecular mass markers are indi-cated on the right (in kDa).

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    Journal of Molecular Neuroscience Volume 22, 2004

    the data presented in Fig. 3. Cystatin C transgeneexpression in the brains of mice belonging to thisline was fivefold higher than mouse endogenouscystatin C levels. Similar levels of both A peptideswere found in the brains of cystatin C transgenicmice compared to nontransgenic littermate controls(Fig. 3). Thus, overexpression of cystatin C in micedoes not affect brain levels of A.

    Growing evidence suggests that cystatin C has arole in AD. First, immunohistochemical studies haverevealed the colocalization of cystatin C with A,predominantly in amyloid-laden vascular walls, butalso in parenchymal amyloid plaques in the brainsof patients with AD and cerebral amyloid angiopa-thy (Maruyama et al., 1990; Vinters et al., 1990; Itohet al., 1993; Haan et al., 1994; Levy et al., 2001), non-demented aged individuals (Levy et al., 2001), agedrhesus and squirrel monkeys (Wei et al., 1996), andtransgenic mice overexpressing human APP(Levy et al., 2001; Steinhoff et al., 2001). Second,immunohistochemical analysis using an anti-cys-tatin C antibody has shown strong punctateimmunoreactivity within the cytoplasm and cellprocesses of pyramidal neurons, mainly in layers IIIand IV of the cortex of aged individuals and ADpatients (Levy et al., 2001). Using an antibody spe-cific to the carboxyl terminus of A42, intracellularimmunoreactivity was observed in the same neu-ronal subpopulation (Levy et al., 2001), suggestingthat A42 accumulates in a specific population ofpyramidal neurons in the brain, the same cell type

    in which cystatin C is highly expressed. Third, colo-calization of cystatin C with APP has been demon-strated in transfected human embryonic kidneyHEK293 cells, mouse neuroblastoma N2a cells(Sastre et al., in press), and in muscle cells of patientswith sporadic inclusion-body myositis (s-IBM) (Vat-temi et al., 2003). Fourth, genetic data have linkedcystatin C gene polymorphisms with late-onset AD(Crawford et al., 2000; Finckh et al., 2000; Beyer etal., 2001; Olson et al., 2002), although some studieswere unable to replicate these findings (Maruyamaet al., 2001; Roks et al., 2001; Dodel et al., 2002). Finally,we recently demonstrated high-affinity binding ofcystatin C to A, which was found to inhibit A fibrilformation (Sastre et al., in press).

    In coimmunoprecipitation experiments we havealso demonstrated binding of cystatin C to full-lengthAPP and to secreted APP. The cystatin C bind-ing domain within APP was localized to the extra-cellular region of A. This binding location seemsto protect APP from -secretase processing,resulting in an increase in the nonamyloidogenic-secretase cleavage, with no effect on the -secretasecleavage site. Accordingly, coexpression of cystatinC and APP in neuroblastoma cells resulted inincreased secretion of APP, whereas productionof both A40 and A42 remained unchanged (Sastreet al., in press). The data presented here demonstratein vivo that overexpression of cystatin C does notaffect the levels of endogenous murine A in thebrain. It remains to be demonstrated whether, sim-ilar to our in vitro observation, cystatin C inhibitsA fibril formation in vivo. To this end, we havecrossed cystatin C transgenic mice with transgenicmice overexpressing APP. Neuropathological andbiochemical examinations of these mice will enablethe in vivo analysis of the effect of cystatin C on Aamyloidogenesis and may help to define the contri-bution of cystatin C to hemorrhage.

    Acknowledgments

    This work was supported by grants from theNational Institute of Neurological Disorders andStroke (NS42029), the National Institute on Aging(AG16837, AG13705, AG08721, and AG17617),American Heart Association (0040102N), and RedCIEN (C03/03) from the Spanish Ministerio deSanidad y Consumo. We thank Dr. Marc Mercken ofJanssen Pharmaceutica and Johnson and JohnsonPharmaceutical Research and Development, for theA antibodies used in the ELISA.

    Fig. 3. Overexpression of cystatin C in transgenic micedoes not affect A production. The concentrations of A40and A42 are presented in fmol/g wet brain as mean S.E.for four transgenic or four nontransgenic mice, 38 mo ofage, with each ELISAmeasurement determined in duplicate.

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    Journal of Molecular Neuroscience Volume 22, 2004

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