©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Apolipoprotein E Carboxyl-terminal Fragments Are Complexed to Amyloids A and L
IMPLICATIONS FOR AMYLOIDOGENESIS AND ALZHEIMER'S DISEASE (*)

(Received for publication, February 21, 1995; and in revised form, May 1, 1995)

Eduardo M. Castao (§) , Frances Prelli , Mordechai Pras (1), Blas Frangione

From the Department of Pathology, New York University Medical Center, New York, New York 10016 Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Apolipoprotein E (ApoE) immunoreactivity is consistently present in the senile plaques and neurofibrillary tangles of Alzheimer's disease (AD) brain. In vitro, apoE, and in particular its apoE4 isoform, can bind to and promote fibrillogenesis of the amyloid A peptide, the main constituent of senile plaques. These findings, together with the strong genetic association between late onset AD and the E4 allele of apoE, have strengthened the hypothesis that apoE may have a central role in the pathogenesis of AD by modulating A cerebral accumulation. However, apoE immunoreactivity is present in all cerebral and systemic amyloidoses tested, and tryptic apoE fragments have been identified in association with amyloid A (AA). In order to further elucidate the interaction between apoE and amyloids, we purified AA and amyloid L (AL) fibrils from patients with familial Mediterranean fever and primary amyloidosis, respectively, and studied the association of apoE with AA and AL proteins. In each case, apoE fragments, detected by Western blot, co-purified with the amyloid fibrils. Microsequencing analysis identified COOH-terminal fragments of apoE, similar to the 10-kDa fragment produced by thrombin digestion that contains the purported binding region to A. In vitro co-incubation of AA with purified human apoE resulted in the formation of an SDS-resistant AAapoE complex and a higher degree of polymerization of the AA peptide. These findings and similar results obtained from AD senile plaques suggest that 1) the carboxyl-terminal fragment of apoE is complexed to amyloid fibrils and resists proteolysis in vivo and 2) apoE may promote amyloidogenesis through a conformation-dependent interaction regardless of the primary structure of the amyloid precursors.


INTRODUCTION

Amyloidosis is a disorder of protein conformation in which low molecular weight proteins that are soluble under physiological conditions become deposited and accumulate either intact or partially digested in diverse tissues and organs as insoluble amyloid fibrils. In spite of their biochemical diversity, amyloid proteins adopt a common secondary structure, the -pleated sheet, and form fibrils of similar morphology. These fibrils are characteristically long straight filaments 5-12 nm wide that share the tinctorial properties of green birefringence after Congo red staining and specific affinity for fluorescent dyes such as Thioflavine S or T. In addition to these features shared by most types of amyloids, there are several factors consistently associated with this pathological condition that can be viewed as part of the biochemical setting in which amyloid deposition arises and develops. These include elevated concentration of amyloid precursors in fluids and tissues and the invariable presence of certain amyloid-associated proteins of which amyloid P-component (AP)()(1, 2) , sulfated proteoglycans (3, 4, 5) , apolipoprotein E (apoE) (6, 7) and apolipoprotein J (8) are the most notorious. Whether these amyloid-associated proteins play an active role by promoting or inhibiting amyloidogenesis or are inert bystanders is at present unknown.

ApoE is a 34-kDa exchangeable apolipoprotein, present in all types of lipoprotein particles, that is involved in cholesterol transport as well as other less defined functions such as nerve regeneration after injury(9, 10, 11, 12) . The recently reported association between certain apoE genetic isoforms and Alzheimer's disease (AD), a condition characterized by the massive deposition in the brain of the 39-44-residue amyloid -protein (A)(13, 14, 15, 16, 17, 18) , has strengthened the hypothesis that this apolipoprotein may be directly involved in the pathogenesis of this cerebral amyloidosis(19, 20) . Moreover, in vitro binding experiments using human apoE isolated from plasma or recombinant human apoE with synthetic peptides homologous to A have shown that these two proteins can form an SDS-resistant complex with A and promote A fibril formation in vitro(21, 22, 23, 24) . These lines of evidence, together with the immunohistochemical colocalization of apoE and A in the senile plaques and its presence in the intraneuronal amyloid of neurofibrillary tangles(6) , suggest that apoE may play an active role in the pathogenesis of AD by promoting fibrillogenesis. However, immunohistochemical studies have shown that apoE is tightly associated with other types of cerebral amyloidosis, including Down syndrome related to A, hereditary cerebral amyloid angiopathy of Icelandic type, related to a cystatin C variant, and spongiform encephalopathies such as Creutzfeldt-Jakob, kuru, and Gerstmann-Sträussler-Scheinker disease that are associated with the prion amyloid(6, 7, 25) . Furthermore, apoE has been immunohistochemically identified within amyloid deposits in systemic forms of the disease such as secondary amyloidosis and familial Mediterranean fever related to amyloid A, immunoglobulin-related primary amyloidosis (AL), and familial amyloidotic polyneuropathy due to deposition of transthyretin genetic variants(7) . Noteworthy, apoE is rarely detected in the nonfibrillar monoclonal deposits of light chain deposition disease and light and heavy chain deposition disease, which may be considered preamyloid forms of AL disease(26) .

This widespread association of apoE with biochemically and clinically diverse types of amyloidoses suggests that apoE may participate in a general manner in the process of amyloid formation. In order to gain insight into the biochemistry of the association between apoE and amyloid proteins, we have characterized by Western blot and amino-terminal sequence analysis apoE fragments that co-purified with amyloid subunits from cases of systemic amyloidosis AA and AL. We also studied the in vitro binding of human apoE isolated from plasma to these amyloid proteins.


EXPERIMENTAL PROCEDURES

Protein Purification

Amyloid A and AL fibrils were isolated by the method of Pras et al.(27) from spleen tissue of patients with familial Mediterranean fever (COH) and primary amyloidosis (RAM). Briefly, 20 g of spleen tissue were homogenized in 0.15 M sodium chloride and centrifuged at 8,000 g for 30 min at 4 °C, and the supernatant was discarded. This procedure was repeated until the supernatant had an absorbance of less than 0.075 at 280 nm. Then the insoluble residue was homogenized in distilled water and centrifuged at 8,000 g at 4 °C for 1 h. The amyloid fibrils that appear as a mucoid mass in the upper layer were dialyzed against water and lyophilized. After lyophilization, AA protein was purified on high performance liquid chromatography (HPLC) (see below). AL fibrils were solubilized in 3 ml of 6 M guanidine hydrochloride, 0.1 M Tris, 0.17 M dithiothreitol, pH 10.2 and stirred for 48 h at room temperature. Then 1 ml of 2 M guanidine hydrochloride, 4 M acetic acid was added, and the solution was applied to a column (2.5 180 cm) consisting of 1:1 (wt) Sephadex G-75 and Sephadex G-100 (Pharmacia Biotech Inc.), equilibrated with 5 M guanidine hydrochloride, 1 M acetic acid. AL amyloid fragments isolated by size exclusion chromatography and AA fibrils were subjected to reverse phase chromatography on a Deltapak C column (0.78 30 cm, Waters) with a gradient of 30-80% acetonitrile in 0.1% (v/v) trifluoroacetic acid, pH 2.5. The column eluents were monitored at 214 nm, and protein peaks were pooled and lyophilized.

Crude amyloid fibrils and fractions obtained by gel filtration and HPLC were subjected to 12.5% Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(28) , and the proteins were electrophoretically transferred (1 h, 400 mA, 4 °C) to polyvinylidene difluoride membranes (Immobilon, Millipore) using 10 mM CAPS buffer, pH 11, containing 10% methanol. The membranes were blocked with 5% nonfat dry milk in 10 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween 20, pH 7.6, for 2 h at room temperature and then incubated overnight at 4 °C with the following antibodies: rabbit polyclonal antibodies to amyloid A(29) , light chain (Dako Corp.), goat anti-human apolipoprotein E (Fitzgerald), and monoclonal antibodies ID7 and 6C5 raised against residues 142-158, and 1-15 of apoE, respectively (a generous gift of Dr Y. Marcel). Horseradish peroxidase-conjugated sheep anti-mouse (Amersham Corp.), goat anti-rabbit, and rabbit anti-goat IgG (Biosource International) were used as the second antibody at a dilution of 1:5000. Immunoblots were visualized with an ECL chemiluminescence kit (Amersham Corp.), according to the manufacturer's specifications. Anti-apoE antiserum was adsorbed by incubating 15 µg of purified human apoE with anti-apoE diluted 1:100 in 0.3% bovine serum albumin (BSA) in Tris-buffered saline, pH 7.4, for 3 h at room temperature.

Protein Sequence Analysis

Automated Edman degradation sequence analysis was carried out on a 477A Protein/Peptide Sequenator, and the resulting phenylthiohydantoin amino acid derivatives were identified using the on-line 120A PTH Analyzer (Applied Biosystems, Foster City, CA).

In Vitro Incubation of ApoE and Amyloid Peptides

Human ApoE was purchased from Cortex Biochem, and bovine ubiquitin and BSA were purchased from Sigma. The purity (> 95%) of these proteins was assessed by SDS-PAGE and NH-terminal sequence analysis. Since human pooled apoE was used, it seems likely that its major isoform is apoE3 according to the statistical distribution in the normal population. Stock solutions of amyloid peptides were prepared in 0.1% trifluoroacetic acid, 50% acetonitrile and quantitated by amino acid analysis using a Pico-Tag analyzer (Waters) or by using a microbicinchoninic acid assay kit (Pierce). ApoE stock solution was made at 0.7 mg/ml in 0.1 M Tris, pH 7.4, and aliquots were stored at -20 °C. Aliquots from these stock solutions were lyophilized and used for the co-incubation experiments as described(21) . In brief, 0.5 µg of apoE were incubated with 10 µg of AA or 15 µg of AL in 12 µl of 0.1 M Tris, pH 7.4, for the indicated time at room temperature. After incubation, 15 µl of 4% SDS-sample buffer were added, and the mixture was run on 12.5% SDS-Tricine gels or on nondenaturing 7.5% polyacrylamide gels without SDS. Proteins were transferred to Immobilon P and detected using polyclonal anti-apoE or anti-AA and anti-AL antibodies as described above. As a control for the co-incubation experiments, ubiquitin (molecular mass, 8 kDa) and BSA were used instead of amyloid peptides and apoE, respectively.

Immunohistochemistry

Sections of spleen tissue from cases of amyloid A (COH) and AL (RAM) were deparaffinized in xylene and rehydrated in ethanol and Tris-buffered saline. After rehydration, endogenous peroxidase was quenched by incubation with 0.3% hydrogen peroxide in methanol for 30 min. After blocking with 3% BSA in Tris-buffered saline, sections were incubated with goat anti-human apoE (Fitzgerald) at a 1:100 dilution overnight at 4 °C and rabbit anti-goat labeled with horseradish peroxidase (Biosource International) at 1:1000 for 1 h. The reaction was detected using 0.03% 3,3`-diaminobenzidine tetrachloride and 0.01% hydrogen peroxide, 50 mM Tris, pH 7.4. Anti-apoE antiserum was adsorbed with purified human apoE as described above.


RESULTS

Immunohistochemistry of spleen sections from patients COH and RAM showed that polyclonal anti-human apoE labeled amyloid deposits as described previously(7) . This immunoreactivity was completely abolished after adsorption with purified human apoE, indicating its specificity (Fig. 1).


Figure 1: Immunohistochemistry on spleen sections of patient COH with amyloid A. a, anti-apoE antibody labels amyloid-laden vessels. The bar represents 20 µm. b, an adjacent section as in a after adsorption of anti-apoE with human purified apoE.



Amyloid L from patient RAM was purified by the procedure of saline-water extraction and gel chromatography on Sephadex G-100 equilibrated with 5 M guanidine. SDS-PAGE of the fractions revealed that the main amyloid subunit had a molecular mass of 12-13 kDa, and amino-terminal sequence analysis showed its homology to a III or IV Ig light chain (Fig. 3). HPLC separation of RAM AL yielded a broad peak between 45 and 73% solvent B. This peak was divided into four fractions, A, B, C, and D, which were rechromatographed (Fig. 2a).


Figure 3: Microsequence Analysis. Amino-terminal sequence of amyloid L () and amyloid A () proteins and of the apoE peptides that co-purified with each of them.




Figure 2: Purification of RAM Amyloid L. a, reverse phase HPLC of RAM amyloid L on a Deltapak C column (0.78 30 cm) using a 30-80% linear gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid. RAM amyloid L eluted as a broad peak between 45 and 73% of solvent B. b, Coomassie Blue stain of 12.5% Tricine-SDS-PAGE of RAM AL fibrils (F) and of HPLC fractions A, B, C, and D. RAM amyloid L subunit has a mass of 12-13 kDa (K) and is present in all the fractions together with a minor dimeric component of 26 kDa. The 8-9-kDa band present in fraction C is a carboxyl-terminal truncated fragment of AL. c, Western blot analysis of HPLC fractions A, B, C, and D using an antibody to human apoE and developed with chemoluminescence showed a major 8-9-kDa band and a minor 18-kDa band in fraction D. The approximate yield of the apoE fragment obtained from RAM amyloid was estimated as follows. The ratio of amyloid to apoE in fraction D was determined by densitometry of the Coomassie Blue-stained 12-13-kDa and 26-kDa AL bands and the 8-9 kDa apoE band in a PDI optical densitometer. This ratio was applied to the area of fraction D in the HPLC profile. Then the proportional area of fraction D to the total area of the HPLC profile was determined.



SDS-PAGE analysis of these fractions revealed that in addition to the major AL subunit of 12-13 kDa, a 26-kDa component was seen in all the fractions. The latter probably corresponded to a dimer of AL subunit since it was recognized by anti- chain antibody (not shown). In addition, minor bands of approximately 8-9 kDa were present in fractions C and D (Fig. 2b). Western blot analysis of the same fractions using anti-apoE antibody and visualized by chemiluminescence detection revealed a major band at 8-9 kDa and a minor 24-kDa component in fraction D (Fig. 2c). The failure to detect apoE by direct amino-terminal sequence analysis of RAM fraction D, despite the positive apoE immunoreactivity of this fraction, indicated that it was present in a very low concentration. Therefore, the HPLC fractions A, B, C, and D were run on SDS-Tricine gels and transferred to Immobilon membrane, and the bands were excised and subjected to amino-terminal sequence analysis. Amino-terminal sequence of the 8-9-kDa component of fraction C revealed that it was a fragment of AL starting at position 1 (not shown). The 8-9-kDa band of HPLC fraction D yielded two major fragments of apoE starting at positions 225 and 227 and a minor one starting at position 216 in addition to the amyloid protein (Fig. 3). The estimated relative yield of apoE carboxyl-fragment extracted from these amyloid deposits was approximately a 1:50 molar ratio, apoE:amyloid.

Purification of amyloid A subunits from COH was performed by saline-water extraction followed by separation on HPLC (Fig. 4a). This procedure yielded broad ill defined peaks that eluted at 60-78% solvent B, which were pooled into four fractions, A, B, C, and D, that were rechromatographed and analyzed by SDS-PAGE. A major protein band of 6-8 kDa was present in all the fractions, and a minor 16-kDa component, which may be a polymer of the 6-8-kDa peptide, was detected in fractions B and C (Fig. 4b). Amino-terminal sequence analysis of the HPLC fractions revealed only one residue per cycle. The sequence was identical to serum amyloid A protein (Fig. 3). No additional sequences were identified. When the same fractions were subjected to Western blot using anti-apoE antibody, the fluorogram revealed a broad band at 8-9 kDa in fraction D (Fig. 4c). Immunoreactivity of this band was completely suppressed after adsorption of the antiserum with purified human apoE, reflecting the specificity of the reaction. There was no band corresponding to intact apoE (34 kDa), suggesting that only small fragments of apoE were associated with the amyloid A subunit.


Figure 4: Purification of COH amyloid A proteins. a, reverse phase HPLC of COH amyloid A on a Deltapak C column (0.78 30 cm) using a 30-80% linear gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid. COH amyloid A eluted as a broad peak between 60 and 78% of solvent B. b, 12.5% Tricine-SDS-PAGE of HPLC fractions A, B, C, and D. A major COH amyloid A subunit of 6-8 kDa (K) was present in all the fractions. A minor 16-kDa component, possibly a polymer of the former, was detected in fractions B and C. c, Western blot analysis of HPLC fractions A, B, C, and D using an antibody to human apoE and developed by chemiluminescence revealed a broad 8-9-kDa component in fraction D.



The failure to detect apoE by direct amino-terminal sequence analysis of COH fraction D, despite the positive apoE immunoreactivity, indicated that it was present in a very low concentration. Therefore, a similar approach as for RAM AL was used, and proteins were separated on SDS-Tricine gels and transferred to Immobilon membrane. The upper portion of the 6-8-kDa band of HPLC fraction D yielded two sequences corresponding to the N terminus of amyloid A and to an apoE fragment starting at position 199, respectively (Fig. 3). In order to investigate whether intact apoE was present in the crude A and L amyloid fibril fraction before HPLC separation, we performed Western blots using monoclonal antibodies ID7 and 6C5, specific for the amino-terminal domain of apoE. A band corresponding to the 34-kDa intact apoE was observed only in association with COH fibrils.

When 0.5 micrograms of purified human apoE were incubated with 10 µg of purified COH amyloid A for 6 h at room temperature in 0.1 M Tris, pH 7.4, a novel component of approximately 44 kDa was detected by Western immunoblot analysis using anti-apoE (Fig. 5a) and anti-AA antibodies (not shown), indicating that an apoE-amyloid complex partially resistant to SDS was formed. Under nondenaturing conditions in 7.5% polyacrylamide gels, this complex resulted in a shift of the electrophoretic mobility of apoE (Fig. 5b). No complex formation was detected when apoE was incubated with 20 µg of ubiquitin. The influence of apoE upon AA polymerization was evaluated by detection of the apoE-AA incubation mixture with an anti-AA antibody on Western blots. AA alone incubated for 6 h at room temperature in 0.1 M Tris, pH 7.4, showed mainly monomeric forms and minor polymeric components after SDS-PAGE. In contrast, AA co-incubated with apoE at a 1:85 molar ratio (apoE:amyloid) under the same conditions as above resulted in the appearance on the gel of a higher amount of AA dimers, trimers, tetramers, and higher molecular weight components (Fig. 5c). AA co-incubated with BSA showed no increment in polymerization when compared with AA alone (not shown). When the same experiment was done using RAM AL, no additional bands were present. However, most of the amyloid-apoE incubation mixture remained on top of the stacking and running gels as very high molecular weight components (not shown). This result is consistent with a higher degree of aggregation of amyloid AL subunits in the presence of apoE that resisted denaturation by SDS treatment.


Figure 5: In vitro co-incubation of amyloid A with apoE. a, ApoE (0.5 µg) and amyloid A (10 µg) were incubated in 0.1 M Tris, pH 7.4, for 6 h at room temperature. The incubation was stopped by the addition of 2 Laemmli sample buffer, and samples were run on 12.5% Tricine SDS-PAGE. After transferring to Immobilon P, proteins were detected with anti-apoE antibody. Lane1, apoE alone; lane2, apoE incubated with amyloid A. Leftmargin indicates molecular mass in kilodaltons (kD). E, apoE; EA, apoE-amyloid A complex. b, apoE and amyloid A were incubated as described above and run on nondenaturing 7.5% polyacrylamide gels. Proteins were detected on Western blots with anti-apoE antibody. A shift in the electrophoretic mobility of apoE reflects the formation of an amyloid-apoE complex. Lane1, apoE alone; lane2, apoE incubated with amyloid A. c, amyloid A was incubated alone or with apoE as described above. Samples were run on 12.5% SDS-Tricine PAGE and transferred to Immobilon P membranes. Proteins were detected with anti-AA antibody. A higher degree of polymerization of amyloid A peptide was present upon incubation with apoE. Lane1, amyloid A alone; lane2, amyloid A incubated with apoE. Leftmargin indicates molecular mass in kDa.




DISCUSSION

Apo E is a two-domain protein, modeled by its two major fragments after thrombin digestion. The 22-kDa amino-terminal domain (residues 1-191) is a stable globular structure containing the sequence that mediates low density lipoprotein-receptor binding(9, 30) . In contrast, the 10-kDa carboxyl-terminal fragment is less stable, binds to lipoproteins, and mediates lipid-free apoE tetramerization in aqueous solutions(31) . Previously, we published the finding of a tryptic fragment of apolipoprotein E corresponding to residues 270-278 in association with two amyloid A proteins(29) . Our present data extend that observation. Three fragments of apoE, located within the thrombolytic carboxyl-fragment of apoE, co-purified with RAM amyloid L. A slightly larger fragment of apoE starting in the region connecting the thrombolytic amino-terminal and carboxyl-terminal domains of apoE co-purified with COH amyloid A (Fig. 6). Similar apoE fragments have been recently obtained from AD senile plaques (32


Figure 6: Schematic representation of the structural domains of apolipoprotein E (a) modeled by thrombin proteolytic cleavage(31) . Alignment of the apoE carboxyl-terminal peptides associated with AL and AA proteins is shown as well as a representation of the purported apoE ``binding domain'' for A (b)(20) .



We propose that the apoE fragments that co-purify with AA and AL are bound to these amyloid proteins in vivo. The remainder of the apoE molecule is cleaved by thrombin and/or other serine proteases in situ either before or after the amyloid-apoE complex is formed. Alternatively, we cannot rule out that apoE was partially degraded during purification. Intact apoE and apoE fragments, as determined by Western blot analysis, are present in the crude AA and AL fibril fractions before HPLC. However, after purification only the carboxyl-terminal apoE fragment co-purifies with the AA and AL amyloids. The question of whether the apoE-amyloid association precedes tissue deposition remains to be addressed.

It is likely that the initial formation of stable apoE-amyloid complexes requires the interaction of the amino and carboxyl-terminal domains of apoE. Binding to the low density lipoprotein receptor through the amino-terminal domain of apoE requires lipid association, largely mediated by the carboxyl end of the molecule(31) . The interdependence of the domains of apoE is further reflected by the effect that amino acid substitutions within the amino-terminal region have on the association of apoE with diverse lipoprotein classes. Such association is known to be mediated by the carboxyl terminus of apoE (9) . The apoE3 isoform with a Cys at position 112 preferentially binds high density lipoproteins, whereas the presence of Arg at the same position favors binding to very low density and intermediate density lipoproteins(33, 34) . A cooperation between domains unique to each apoE isoform has been postulated to explain this selective lipoprotein distribution(35) . Whether a similar mechanism can account for the higher avidity of the apoE4 isoform for A of AD remains to be tested.

ApoE is capable of binding to synthetic A through a strong interaction resistant to SDS(21) . A similar interaction between apoE and A of the Dutch variant of cerebral amyloid angiopathy, a rare form of familial AD, has also been documented (36) (details of apoE-A binding will be published elsewhere). By using a set of truncated recombinants, the purported binding region to A has been located within the carboxyl-end of apoE between amino acids 244 and 272 (21) . Our findings suggest that the binding of this region to A is not specific for this peptide but rather reflects a hydrophobic interaction between apoE carboxyl end and a common conformation shared by different amyloids. Secondary structure predictions indicate that a putative amphipathic helix with high affinity for lipids (37) is located within the region that co-purifies with amyloid peptides. Possibly, other exchangeable apolipoproteins sharing similar amphipathic helices can also have amyloid-apolipoprotein interactions. It is noteworthy that genetic variants of apoAI can form amyloid in certain hereditary forms of human amyloidosis (38, 39) and that SAA, the precursor of AA, is itself an apolipoprotein associated to high density lipoprotein(40) . We recently found that apoAI can bind to A and promote A fibrillogenesis in vitro.()Presumably, in the same way that there is a large number of amyloidogenic precursor proteins that can end as insoluble amyloid fibrils, there is also a group of amyloidogenic apolipoproteins and acute phase reactants that can actively participate in this process either as constituents of fibrils or as modulators of fibrillogenesis.

Our finding of a higher degree of polymerization of AA and AL native peptides upon incubation with apoE together with previous work using synthetic A (22, 23, 24, 36) points to a general role of apoE in amyloidogenesis. The molar ratios of apoE and AA used in our in vitro experiments are opposite of those found in plasma in normal conditions for apoE and SAA. However, SAA is an acute phase reactant, and its concentration in plasma is capable of rising 1000-fold during tissue injury or inflammation(41) . AL, in turn, is derived from monoclonal light chains, which are known to have very high levels in the circulation in most of the cases. Therefore, we believe that the concentrations that we used in vitro can resemble more closely the pathological conditions in which amyloidosis develops than the physiological levels of apoE and amyloid precursors.

ApoE may act as one of a group of pathological chaperones that promote the aggregation of amyloidogenic precursors of diverse primary structure into the -pleated sheet conformation of amyloid fibrils. The absence of apoE and AP in the nonfibrillar, Congo red negative monoclonal deposits of light chain deposition disease and light and heavy chain deposition disease, which are considered preamyloid forms of AL disease(26) , suggests that apoE and AP may be essential catalysts for the modulation of the amyloid generation process. The interaction between apoE and amyloids may also be influenced by other local acute phase reactants such as proteoglycans to which both amyloid precursors and apoE are known to bind(42, 43) . In spite of this putative widespread role of apoE in amyloid formation, it seems likely that a certain specificity exists between some of the amyloid precursors and apoE genetic isoforms. Thus far, no preferential associations between systemic amyloidoses and apoE isoforms have been reported; however, the genetic association of AD with apoE4 and the in vitro higher avidity of this isoform for A may reflect such specificity(19, 21) . Alternatively, the apoE4 isoform may participate in the pathogenesis of AD through a different pathway unrelated to A formation or deposition.

Recently it has been reported that apoE can inhibit A aggregation in vitro at concentrations similar to those found normally in biological fluids(44) . This apparent discrepancy with previous studies could be due to different experimental conditions(22, 23, 24, 36) . However, it raises the intriguing possibility that apoE may have a dual effect upon fibrillogenesis in vivo depending on the local concentrations of both apoE and amyloid precursors in sites of amyloid formation. Within physiological levels, apoE may have a protective role on amyloid formation by sequestering soluble amyloid precursors, as has been postulated for A(44) . However, A appears to be associated to other apolipoproteins (i.e. apolipoprotein J) in biological fluids in vivo(45) . Yet, in tissues in which local membrane repair and lipid turnover are increased, such as after cell injury, apoE is overexpressed and can reach higher concentrations than those found normally in the circulation(10, 11, 12, 46) . Under these circumstances, apoE may have a substantially different effect upon its association with amyloid precursors. In this pathological setting, several other factors may affect apoE behavior, such as its state of oxidation, lipid association, the presence of other amyloid/apoE binding proteins, and the stage of the amyloid process(47) .

The abnormal amyloid response in each individual will be determined finally by a complex process involving critical concentrations of amyloid precursors and a set of amyloid-associated proteins such as apoE, other apolipoproteins, AP, -antichymotrypsin (22) , and proteoglycans. These may act in conjunction with yet undefined tissue-specific factors to modulate the conformational transition of a soluble protein into an insoluble fibril. A better understanding of these complex interactions between amyloids and the factors that modulate their formation may open novel strategies for the treatment of the systemic and cerebral amyloid-related diseases.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR02594 (MERIT), AG10953 (LEAD), and AG05891 and the Metropolitan Life Foundation Award for Medical Research, 1993. Part of this work was presented at the 1994 Annual Meeting of the Society for Neuroscience, Miami Beach, Florida. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, New York University Medical Center, 550 First Ave., TH427, New York, NY 10016. Tel.: 212-263-5775; Fax: 212-263-6751.

The abbreviations used are: AP, amyloid P-component; HPLC, high performance liquid chromatography; AL, amyloid L; SAA, serum amyloid A precursor; AA, amyloid A; AD, Alzheimer's disease; CAPS, 3-(cyclohexylamino)propanesulfonic acid; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

E. M. Castao, unpublished observations.


REFERENCES
  1. Coria, F., Castao, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L., and Frangione, B.(1988)Lab. Invest. 58, 454-458 [Medline] [Order article via Infotrieve]
  2. Duong, T., Pommier, E. C., and Scheibel, A. B.(1989)Acta Neuropathol. 78, 429-437 [Medline] [Order article via Infotrieve]
  3. Snow, A. D., Willmer, J., and Kisilevsky, R.(1987)Lab. Invest. 56, 120-123 [Medline] [Order article via Infotrieve]
  4. Snow, A. D., Nochlin, D., Sumi, S., Bird, T. D., and Wight, T. N.(1988) Alzheimer Dis. Assoc. Disord. 2, 232-240
  5. Young, I. D., Willmer, J. P., and Kisilevsky, R.(1989)Acta Neuropathol. 78, 202-209 [Medline] [Order article via Infotrieve]
  6. Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E., and Ikeda, K.(1991)Brain Res. 541, 163-166 [CrossRef][Medline] [Order article via Infotrieve]
  7. Wisniewski, T., and Frangione, B.(1992)Neurosci. Lett. 135, 235-238 [CrossRef][Medline] [Order article via Infotrieve]
  8. Choi-Miura, N. H., Ihara, Y., Fukuchi, K., Takeda, M., Nakano, Y., Tobe, T., and Tomita, M. (1992)Acta Neuropathol. 83, 260-264 [Medline] [Order article via Infotrieve]
  9. Mahley, R. W. (1988)Science 240, 622-630 [Medline] [Order article via Infotrieve]
  10. Boyles, J. K., Zoellner, C. D., Anderson, L. J., Kosik, L. M., Pitas, R. E., Weisgraber, K. H., Hui, D. Y., Mahley, R. W., Gebicke-Haerter, P. J., Ignatius, M. J., and Shooter, E. M. (1989) J. Clin. Invest.83, 1015-1031 [Medline] [Order article via Infotrieve]
  11. Leblanc, A. C., and Poduslo, J. F.(1990)J. Neurosci. Res. 25, 162-171 [Medline] [Order article via Infotrieve]
  12. Poirer, J., Bacchichet, A., Dea, D., and Gauthier, S.(1993)Neurosci. 55, 81-90 [CrossRef][Medline] [Order article via Infotrieve]
  13. Glenner, G. G., and Wong, C. W.(1984)Biochem. Biophys. Res. Commun. 122, 1131-1135 [Medline] [Order article via Infotrieve]
  14. Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985)Proc. Natl. Acad. Sci. U. S. A. 82, 4245-4249 [Abstract]
  15. Wong, C. W., Quaranta, V., and Glenner, G. G.(1985)Proc. Natl. Acad. Sci. U. S. A. 82, 8729-8732 [Abstract]
  16. Miller, D. L., Papayannopoulos, I. A., Styles, J., Bobin, S. A., Lin, Y. Y., Biemann, K., and Iqbal, K.(1993)Arch. Biochem. Biophys. 301, 41-52 [CrossRef][Medline] [Order article via Infotrieve]
  17. Selkoe, D. J., Abraham, C. R., Podlisny, M. B., and Duffy, L. K.(1986)J. Neurochem. 146, 1820-1834
  18. Wisniewski, T., Lalowski, M., Levy, E., Marques, M. A., and Frangione, B.(1994) Ann. Neurol. 35, 245-246 [Medline] [Order article via Infotrieve]
  19. Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., and Pericak-Vance, M. A.(1993) Science 261, 921-923 [Medline] [Order article via Infotrieve]
  20. Strittmatter, W. J., Saunders, A. M., Schmechel, D., Pericak-Vance, M., Enghild, J., Salvesen, G. S., and Roses, A. D.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 1977-1981 [Abstract]
  21. Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L. M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 8098-8102 [Abstract/Free Full Text]
  22. Ma, J., Yee, A., Brewer, B., Das, S., and Potter, H.(1995)Nature 372, 92-94
  23. Wisniewski, T., Castao, E. M., Golabek, A., Vogel, T., and Frangione, B.(1994) Am. J. Pathol. 145, 1030-1035 [Abstract]
  24. Sanan, D. A., Weisgraber, K. H., Russell, S. J., Mahley, R. W., Huang, D. Y., Saunders, A., Schmechel, D, Wisniewski, T., Frangione, B., Roses, A. D., and Strittmatter, W. J. (1994) J. Clin. Invest.94, 860-869 [Medline] [Order article via Infotrieve]
  25. Bugiani, O., Giaccone, G., Frigerio, L., Farlow, M. R., Ghetti, B., and Tagliavini, F. (1994)Neurobiol. Aging15,S156-157
  26. Gallo, G., Wisniewski, T., Choi-Miura, N-H., Ghiso, J., and Frangione, B.(1994) Am. J. Pathol. 145, 526-530 [Abstract]
  27. Pras, M., Schubert, M., Zucker-Franklin, D., Rimon, A., and Franklin, E. C.(1968) J. Clin. Invest. 47, 924-933 [Medline] [Order article via Infotrieve]
  28. Schägger, H., and von Jagow, G.(1987)Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  29. Prelli, F., Pras, M., Shtrasburg, S., and Frangione, B.(1991)Scand. J. Immunol. 33, 783-786 [Medline] [Order article via Infotrieve]
  30. Wilson, C., Wardell, M. R., Weisgraber, K. H., Mahley, R. W., and Agard, D. A.(1991) Science 252, 1817-1822 [Medline] [Order article via Infotrieve]
  31. Wetterau, J. R., Aggerbeck, L. P., Rall, S. C., Jr., and Weisgraber, K. H.(1988) J. Biol. Chem. 263, 6240-6248 [Abstract/Free Full Text]
  32. Wisniewski, T., Lalowski, M., Golabek, A., Vogel, T., and Frangione, B.(1995) Lancet 345, 956-958 [CrossRef][Medline] [Order article via Infotrieve]
  33. Gregg, R. E., Zech, L. A., Schaefer, E. J., Stark, D., Wilson, D., Brewer, H. B., Jr. (1986)J. Clin. Invest. 78, 815-821 [Medline] [Order article via Infotrieve]
  34. Steinmetz, A., Jakobs, C., Motzny, S., and Kaffarnik, H.(1989) Arteriosclerosis 9, 405-411 [Abstract]
  35. Weisgraber, K. H. (1990)J. Lipid Res. 31, 1503-1511 [Abstract]
  36. Castao, E. M., Prelli, F., Wisniewski, T., Golabek, A., Kumar, R. A., Soto, C., and Frangione, B.(1995)Biochem. J. 306, 599-604 [Medline] [Order article via Infotrieve]
  37. Segrest, J. P., Jones, M. K., De Loof, H., Brouillette, C. G., Venkatachalapathi, Y. V., and Anantharamaiah, G. M.(1992)J. Lipid. Res. 33, 141-166 [Abstract]
  38. Nichols, W. C., Dwulet, F. E., Liepnieks, J., and Benson, M. D.(1988) Biochem. Biophys. Res. Commun. 156, 762-768 [Medline] [Order article via Infotrieve]
  39. Soutar, A. K., Hawkins, P. N., Vigushin, D. M., Tennent, G. A., Booth, S. E., Hutton, T., Nguyen, O., Totty, N. F., Feest, T. G., Hsuan, J. J., and Pepys, M. B. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 7389-7393 [Abstract]
  40. Benditt, E. P., Erikssen, N., and Hanson, R. H.(1979)Proc. Natl. Acad. Sci. U. S. A. 76, 4092-4096 [Abstract]
  41. McAdam, K. P. W. J., and Sipe, J. D.(1976)J. Exp. Med. 144, 1121-1127 [Abstract]
  42. Stenstad T., Magnus, J. H., Syse, K., and Husby, G.(1993)Clin. Exp. Immunol. 94, 189-195 [Medline] [Order article via Infotrieve]
  43. Weisgraber, K. H., Rall, S. C., Jr., Mahley, R. W., Milne, R. W., Marcel, Y. L, and Sparrow, J. T.(1986)J. Biol. Chem. 261, 2068-2076 [Abstract/Free Full Text]
  44. Evans, K. C., Berger, E., Cho, C. G., Weisgraber, K. H., and Lansbury, P. T., Jr. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 763-767 [Abstract]
  45. Ghiso, J., Matsubara, E., Koudinov, A., Choi-Miura, N-H., Tomita, M., Wisniewski, T., and Frangione, B.(1993)Biochem. J. 293, 27-30 [Medline] [Order article via Infotrieve]
  46. Snipes, G. J., McGuire, C. B., Norden, J. J., and Freeman, J. A.(1986)Proc. Natl. Acad. Sci. U. S. A. 83, 1130-1134 [Abstract]
  47. LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994)J. Biol. Chem. 269, 23403-23406 [Abstract/Free Full Text]

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