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(Stroke. 1996;27:2080-2085.)
© 1996 American Heart Association, Inc.

Articles

Cystatin C

Icelandic-Like Mutation in an Animal Model of Cerebrovascular ß-Amyloidosis

LiHong Wei, MD; Lary C. Walker, PhD Efrat Levy, PhD

the Departments of Pharmacology (E.L., L.W.) and Pathology (E.L.), New York University Medical Center, New York, NY; and the Department of Neuroscience Pharmacology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert, Ann Arbor, Mich (L.C.W.).

Correspondence to Efrat Levy, PhD, New York University Medical Center, 550 First Ave, MSB249, New York, NY. E-mail levye01@mcrcr.med.nyu.edu.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 
Background and Purpose Cerebral amyloid angiopathy (CAA) occurs as a sporadic disorder in aged humans, as a frequent component of Alzheimer's disease, or in hereditary cerebral hemorrhage with amyloidosis (HCHWA). The primary histological locus of cerebral amyloid deposition varies in aged humans and in different species of nonhuman primates. In aged rhesus monkeys, amyloid deposition occurs most frequently in senile plaques, whereas in aged squirrel monkeys CAA is more common. We hypothesized that the preponderance of CAA in squirrel monkeys is related to a species-specific amino acid change in cystatin C, a cysteine protease inhibitor, similar to the Leu68Gln substitution found in the amyloid protein of Icelandic patients with HCHWA-I, also termed hereditary cystatin C amyloid angiopathy.

Methods We performed immunohistochemical analyses of brain sections of aged squirrel and rhesus monkeys with anti–amyloid-ß and anti–cystatin C antibodies and sequenced the cystatin C cDNA of these monkeys.

Results Cerebral amyloid in aged squirrel and rhesus monkeys, previously shown to be immunoreactive with anti–amyloid-ß antibodies, reacts also with antibodies to cystatin C. While the predicted amino acid sequence in rhesus monkeys differs from the human sequence by four residues, that of the squirrel monkeys has seven additional amino acid substitutions, one of which is Leu68Met.

Conclusions The presence of a mutation in squirrel monkeys similar to the one found in HCHWA-I suggests that alterations in cystatin C may influence the likelihood that amyloid will be deposited in the walls of cerebral blood vessels. These observations support the utilization of the monkeys as models to study CAA.


Key Words: aging • Alzheimer's disease • amyloid • cystatins • monkeys


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 
Cerebral amyloid angiopathy (CAA) is an age-associated lesion in which amyloid is deposited primarily in the vascular wall. Patients with CAA display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage, as a result of extensive amyloid deposition within cerebral vessels.1 The amyloid deposited is biochemically heterogeneous. Patients with Alzheimer's disease, Down syndrome, sporadic CAA, and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) have amyloid in cerebral vessel walls, composed mainly of amyloid ß-protein (Aß). Aß is a 39– to 43–amino acid peptide,2 a degradation product of a larger ß-amyloid precursor protein (ßPP).3 Patients with two hereditary forms of CAA have been found to carry mutations in the ßPP gene, resulting in amino acid substitutions in Aß that are implicated as promoters of CAA in these patients.4 5 In the Icelandic autosomal dominant form (HCHWA-I),6 7 also called hereditary cystatin C amyloid angiopathy (HCCAA),8 the amyloid deposited in the cerebral vasculature is a variant of another protein, cystatin C.9 10

Cystatin C11 or {gamma}-trace12 is a basic, low-molecular-weight serum protein found in all body fluids and tissues examined11 12 13 14 15 16 17 and is 5.5 times more concentrated in the cerebrospinal fluid than in plasma.18 It is composed of 120 amino acid residues, with a molecular weight of 13 260 D.19 Cystatin C has the cysteine protease inhibitory property that is shared by the members of the cystatin superfamily including stefins, cystatins, and kininogens.20 21 Comparison of the genes encoding cystatin C, isolated from normal tissue and from the brain of an HCHWA-I patient, revealed their sequence identity except for a single mutation in the Icelandic gene.22 This mutation cosegregated with the disease in every case.23 24 The amyloid protein isolated from the leptomeninges of HCHWA-I patients starts at position 11 of the normal urinary cystatin C and has an amino acid substitution, Leu to Gln, at position 689 10 25 within the segment that contains the purported active site of all known cystatins.20

Immunohistochemical studies of patients with CAA and HCHWA-D, but not HCHWA-I, have demonstrated dual staining of vessels with antibodies to Aß and cystatin C. Colocalization of cystatin C with Aß in cerebrovascular amyloid deposits was most extensive in brains of patients with CAA-related hemorrhage but was also found in amyloidotic microvessels from patients without hemorrhages.26 27 28 29 30 31 32 It was suggested that in these patients cystatin C deposition occurs secondarily to Aß deposition and may play a role in the development of cerebral hemorrhage.

Nonhuman primates are good models for studying age-associated changes in the brain. Several neuropathological changes similar to those present in persons with Alzheimer's disease and normal aged humans have been found in senescent nonhuman primates.33 34 35 The most extensively studied aged primates are rhesus monkeys (Macaca mulatta), Old World primates with a life span of 35 to 40 years,35 and squirrel monkeys (Saimiri sciureus), New World primates with a life span of 25 to 30 years.34 Amyloid deposition in aged rhesus monkeys predominates as senile plaques with relatively minor vascular involvement; however, in aged squirrel monkeys cerebrovascular deposits usually are more conspicuous than are senile plaques.34 36 The amyloid found in the brains of aged squirrel monkeys is associated primarily with intracerebral and meningeal capillaries and arterioles and occurs to a lesser degree as senile plaques.34

We originally hypothesized that a species-specific amino acid difference in Aß or ßPP may contribute to CAA in aged squirrel monkeys. However, the predicted amino acid sequence of the Aß is identical to that in normal humans and rhesus monkeys; overall, ßPP751 in the squirrel monkey differs from the human sequence only by four amino acids near the N-terminus.37 The lack of amino acid differences in the Aß sequence and the overall high degree of homology between squirrel monkey and human ßPP suggest that other factors most likely predispose aged squirrel monkeys to CAA. To determine whether CAA in squirrel monkeys might be related to a specific amino acid substitution in cystatin C, perhaps similar to that found in HCHWA-I, we sequenced the cDNA for cystatin C in squirrel monkeys and rhesus monkeys and compared them with the normal and mutated human forms.


*    Materials and Methods
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 *Materials and Methods
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 down arrowIntroduction 
 
Tissues
For isolation of RNA, tissues (liver or kidney) were removed at necropsy from five squirrel monkeys; two monkeys were young adults and three were aged animals. Brain tissue was obtained from one rhesus monkey. For immunohistochemistry, brain tissues were obtained from seven aged squirrel monkeys (aged 18 to 23 years) and seven aged rhesus monkeys (aged 29 to 33 years). (Brain tissue of one rhesus monkey was kindly provided by Dr Hideo Uno, Wisconsin Regional Primate Research Center.)

Sequence Analysis
Total RNA was purified from frozen tissues by homogenization in 5 mol/L guanidine isothiocyanate, followed by precipitation in 4 mol/L LiCl and then phenol chloroform extraction.38 First strand cDNA synthesis in 40 µL reaction contained 10 µg RNA, 2 µg random hexamer, 46 U RNasin (Promega), 0.1 mol/L dithiothreitol, 0.5 mmol/L dNTPs, and 2 µL SuperScript Reverse transcriptase (Life Technologies) in reverse transcription reaction buffer and was incubated at 37°C for 90 minutes. The reaction was stopped at 65°C and used in the polymerase chain reaction with primers corresponding to sequences flanking the full-length cystatin C sequence, including sequences encoding the signal sequence. The amplification reaction solution (100 µL) contained 4 µL cDNA products, 25 pmol each forward and reverse primers, 200 µmol/L dNTPs, 2 mmol/L MgCl2, and 2.5 U Taq DNA polymerase (Boehringer-Mannheim) in 1x Taq buffer. The samples went through 25 cycles of 94°C for 1 minute, 55°C for 0.5 minute, and 72°C for 1 minute, ending with 72°C for 10 minutes. Amplified fragments were analyzed on polyacrylamide gels, and 1 µL was cloned into pCR II vector (Invitrogen). Sequence analysis of cloned DNA was performed by the dideoxy chain termination method with TaqTrack (Promega).

Immunohistochemistry
Sections of formalin-fixed brains were deparaffinized and treated with 100% formic acid for 10 to 30 minutes to enhance amyloid staining. Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol for 30 minutes. The sections were blocked with phosphate buffer containing 10% fetal calf serum and 0.2% bovine serum albumin for 1 hour, followed by incubation with primary antibodies in the same solution overnight at 4°C. Then the sections were incubated with biotin-conjugated anti-mouse (1:200) or anti-rabbit (1:800) antibodies (Sigma) and with streptavidin–horseradish peroxidase conjugate (1:300) (Amersham), each for 60 minutes at room temperature. Horseradish peroxidase activity was developed with 3,3'-diaminobenzidine with or without 0.005% cobalt hexachloride (Sigma). The following antibodies were used: polyclonal antibody against cystatin C (Axell) (1:800); the same antibody absorbed with cystatin C expressed as glutathione S-transferase fusion protein and immobilized on agarose beads (1:800); rabbit preimmune serum (1:800); and monoclonal antibody to Aß (4G8, Senetek) (1:200).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 
The cystatin C cDNA was sequenced to determine whether CAA in squirrel monkeys might be related to a species-specific amino acid change in the cysteine protease inhibitor. The cystatin C nucleotide sequences of squirrel monkey, rhesus monkey, and human are presented in Fig 1Down. The cystatin C gene encodes a polypeptide of 146 amino acids. The N-terminal 26 amino acids specify a secretory signal sequence. Translation of the obtained sequences revealed that the predicted amino acid sequence in the rhesus monkey differs from the human sequence by four residues. That of the squirrel monkey has seven additional amino acid substitutions, one of which is Leu68Met (Fig 2Down). The amyloid protein isolated from the leptomeninges of Icelandic patients with HCHWA-I is composed of cystatin C containing a single amino acid substitution, Leu68Gln.9 10 25



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  		Figure 1. Nucleotide sequence of human cystatin C cDNA is shown in the second line (hu). Nucleotide substitutions found in squirrel monkey (sm) and in rhesus monkey (rm) compared with the human sequence are indicated in the first and third lines, respectively.



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  		Figure 2. Deduced cystatin C amino acid sequence of squirrel monkey (sm), human (hu), and rhesus monkey (rm). The first 26 amino acids specify a signal sequence. *Amino acid difference. The position of the HCHWA-I polymorphism Leu68Gln is underlined.

Immunohistochemical analysis of brain sections of aged squirrel monkeys and rhesus monkeys with anti–cystatin C antibodies revealed staining of cerebrovascular amyloid deposits and also parenchymal, plaquelike deposits. Staining of adjacent sections with antibodies to Aß demonstrated an abundance of positive cerebrovascular amyloid deposits and a few plaques in aged squirrel monkeys' brains and mainly plaques in brain sections of aged rhesus monkeys. Only some of these amyloid deposits were labeled with anti–cystatin C antibodies (Fig 3Down). These deposits demonstrated a strong reaction with the anti-Aß antibody and a much weaker reaction with the anti–cystatin C antibody (Fig 3Down). Preimmune serum and polyclonal anti–cystatin C antiserum preabsorbed with cystatin C did not label the amyloid, indicating the specificity of the reaction.



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  		Figure 3. Immunohistochemical staining of CAA and senile plaques in aged squirrel monkeys (a through d) and rhesus monkeys (e through h) with polyclonal anti–cystatin C antibody (a, c, e, g) and monoclonal anti–Aß antibody (4G8) (b, d, f, h) (original magnifications: a, b, e, f, x100; c, d, g, h, x400).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 
Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target cysteine endopeptidases by affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. A serine proteinase, elastase, cleaves cystatin C in vitro, generating a very stable product that lacks the N-terminal 10 amino acids of full-length cystatin C, similar to the truncated cystatin C in the amyloid deposits in HCHWA-I patients.9 10 25 There is evidence to indicate that this modification may occur extracellularly in vivo as well; a truncated form of cystatin C was isolated from human urine.39 Isolation of chicken egg white cystatin demonstrated, in addition to the full-length form, shorter forms starting at positions 9 or 10. The shorter forms were shown to be much weaker inhibitors of papain than the full-length forms.40 Truncation of the N-terminal decapeptide of human cystatin C decreased inhibition of papain and human cathepsins B, L, and H.41 42 43 44 45 The N-terminal reactive site was narrowed down to Arg8, Leu9, and Val10.46 47 48 Mutagenesis of N-terminal sequences and in the first and second hairpin loops of the cystatins had different effects on their binding to distinct proteases. It was demonstrated that the decreased affinity of egg white cystatin or human cystatin C for cysteine proteinases is due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme.45 47 Assuming a common mode of interaction with target proteinases for all cystatins, the Gln55-Gly59 and Pro105-Trp106 segments of cystatin C were proposed to be involved in proteinase binding from sequence similarity studies and x-ray crystallography data for chicken cystatin and a cystatin B–papain complex.42 49

The Leu68Met substitution in the squirrel monkey is within the highly conserved region of the cystatin gene family.20 The amyloid protein isolated from the leptomeninges of Icelandic patients with HCHWA-I is composed of cystatin C containing a single amino acid substitution, Leu68Gln.9 10 25 Peptide structure analyses of the cystatin C proteins by the Kyte-Doolittle method reveal a possible increase in the hydrophilicity of the region encompassing the amino acid substitution in the HCHWA-I amyloid subunit. A slight increase in the hydrophilicity of the same region is predicted also for squirrel monkey cystatin C. Chou-Fasman and Garnier-Osguthorpe-Robson methods make secondary structure predictions for a protein sequence.50 51 Both predict an increased {alpha}-helix, N-terminal to the area of the second hairpin loop of the rhesus monkey and squirrel monkey cystatin C compared with that of the human. Of special interest is the Val10Leu substitution in the squirrel monkey cystatin C since this residue was shown to affect the specificity of the inhibitor for different cysteine proteinases.47 The peptide structure predictions suggest that the amino acid substitutions can affect the structure of the proteins. Differences in the inhibitory activity and deposition of human, rhesus monkey, and squirrel monkey cystatin C could result from altered protein structure, substitution of critical active site amino acids, or a combination of both effects.

Several studies have demonstrated that CAA patients with both Aß and cystatin C immunoreactivity in amyloidotic cerebral vessels frequently suffered fatal subcortical hemorrhages, possibly attributable to CAA.26 27 28 29 30 31 In these cases, Aß composed of the normal human sequence forms the amyloid and normal human cystatin C protein is thought to be secondarily deposited. In patients with HCHWA-D, a variant of Aß is predominantly deposited in cerebral vessel walls together with the deposition of normal cystatin C.32 In HCHWA-I patients, a variant of cystatin C forms the amyloid, without any Aß deposition. However, in one hemorrhagic CAA patient not of Icelandic origin, the same variant of cystatin C (Leu68Gln) colocalized with Aß in cerebral vessel walls.52 Although the amyloid deposited in the two HCHWA disorders is biochemically different, the deposition in vessel walls leads to similar clinical phenomena. We propose that specific amino acid substitutions in Aß or cystatin C may modulate massive amyloid deposition in the walls of cerebral blood vessels, weakening the vessel walls and thereby increasing the probability of hemorrhagic stroke. Different substitutions may result in different pathological manifestations. It was previously suggested that the colocalization of both proteins is a fundamental factor in CAA-induced brain hemorrhage in the elderly.28

None of the aged rhesus monkeys tested had evidence of intracerebral hemorrhage; however, colocalization of Aß and cystatin C is demonstrated in the predominant parenchymal deposits as well as vascular amyloid in all of them, with quantitative differences between individual monkeys. Similarly, the amyloid in cerebral vessel walls of all the aged squirrel monkeys is dually stained with anti-Aß antibodies and anti–cystatin C antibodies. While there is no evidence that squirrel monkeys are particularly predisposed to cerebral hemorrhage, no systematic, large-scale study of hemorrhage in aged squirrel monkeys has been conducted. None of the rhesus and squirrel monkeys used in this study died of natural causes, and we cannot rule out the possibility that, given the opportunity to reach older age, they may have developed strokes. One female squirrel monkey known as "Baker" died at the estimated age of 27 years from renal failure. The histopathological analysis of her brain demonstrated abundant amyloid deposition34 and evidence of prior mild hemorrhage (L.G. Wolfe [Auburn University School of Veterinary Medicine] and C.R. Horton, personal communication, 1987).

The processes involving amyloid formation and deposition, the role of amyloid in stroke, and the effect of dual deposition of more than one protein are not known. The specific cystatin C sequences in humans, in rhesus monkeys, and in squirrel monkeys may be responsible for the variability of the amyloid depositions observed. Specific amino acid sequences may affect the binding affinity to other proteins, including cysteine proteinases and possibly Aß. Furthermore, the amino acid substitutions in the squirrel monkey cystatin C may contribute to the predominant deposition of amyloid in cerebral vessel walls. Both species of monkeys are useful models for studying the dual deposition of Aß and cystatin C in the brains of aged individuals as well as the cellular and molecular factors that contribute to CAA in humans.


*    Selected Abbreviations and Acronyms
 
Aß = amyloid ß-protein
ßPP = ß-amyloid precursor protein
CAA = cerebral amyloid angiopathy
HCCAA = hereditary cystatin C amyloid angiopathy
HCHWA-D = hereditary cerebral hemorrhage with amyloidosis, Dutch type
HCHWA-I = hereditary cerebral hemorrhage with amyloidosis, Icelandic type


*    Acknowledgments
 
This study was supported by National Institutes of Health grants AG11481 (Dr Levy) and NS20471 and AG05146 (Dr Walker) from the US Public Health Service. The National Science Foundation supports computing resources through grant BIR-9318128.

Received March 19, 1996; revision received August 8, 1996; accepted August 15, 1996.


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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 

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Editorial Comment

Icelandic-Like Mutation in an Animal Model of Cerebrovascular ß-Amyloidosis

William I. Rosenblum, MD, Guest Editor

Department of Pathology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
 
The accompanying article is relevant to an important question in Alzheimer's disease research, namely, what determines whether ß-amyloid is deposited in senile plaques or in plaques and blood vessel walls. Since many elderly persons have ß-amyloid predominantly in blood vessels and have relatively few senile plaques, the question can be rephrased to ask what determines the predominance of vascular involvement in such persons. This is an important question because congophilic angiopathy caused by ß-amyloid is an important cause of cerebral hemorrhage (lobar hemorrhage) in the elderly.

The authors have compared the brains of aged rhesus monkeys with those of aged squirrel monkeys. They did this because they knew that senile plaques predominate in the rhesus, while congophilic angiopathy predominates in the squirrel monkey. The authors searched for cystatin C in the amyloid deposits because they knew that in one form of human congophilic angiopathy with cerebral hemorrhage an abnormal form of cystatin C is found in the amyloid deposits. The authors demonstrate that a related, abnormal form of cystatin C is present in the vascular amyloid of the squirrel monkeys. The amyloid in the senile plaques of the rhesus also was associated with cystatin C, but this variant of cystatin C lacked the amino acid substitution at the leu68 site that was common to both the human disease (HCHWA-I) and the vascular deposits of squirrel monkeys.

The authors conclude that this abnormal cystatin C may be responsible for the increased deposition of amyloid in cerebral blood vessels of squirrel monkeys. It remains to be seen whether an abnormal cystatin C is also related to ß-amyloid deposition in aged humans generally and whether it is the amount of amyloid, the presence of cystatin C, or both that determines the susceptibility to hemorrhage.


*    Selected Abbreviations and Acronyms
 
Aß = amyloid ß-protein
ßPP = ß-amyloid precursor protein
CAA = cerebral amyloid angiopathy
HCCAA = hereditary cystatin C amyloid angiopathy
HCHWA-D = hereditary cerebral hemorrhage with amyloidosis, Dutch type
HCHWA-I = hereditary cerebral hemorrhage with amyloidosis, Icelandic type




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