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Originally published In Press as doi:10.1074/jbc.M407283200 on October 15, 2004

J. Biol. Chem., Vol. 279, Issue 53, 56004-56013, December 31, 2004
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Insulin-degrading Enzyme in Brain Microvessels

PROTEOLYSIS OF AMYLOID {beta} VASCULOTROPIC VARIANTS AND REDUCED ACTIVITY IN CEREBRAL AMYLOID ANGIOPATHY*

Laura Morelli{ddagger}, Ramiro E. Llovera{ddagger}, Irina Mathov{ddagger}, Lih-Fen Lue§, Blas Frangione¶, Jorge Ghiso¶, and Eduardo M. Castaño{ddagger}||

From the {ddagger}Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Química Biológica Patológica, Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, C1113AAD, Buenos Aires, Argentina, the §Sun Health Research Institute, Sun City, Arizona 85351, and the Departments of Pathology and Psychiatry, New York University School of Medicine, New York, New York 10016

Received for publication, June 29, 2004 , and in revised form, September 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The accumulation of amyloid {beta} (A{beta}) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the A{beta} sequence. A higher tendency of A{beta} to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to A{beta} accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading 125I-insulin and cleaved A{beta}-(1-40) wild type and the genetic variants A{beta} A21G (Flemish), A{beta} E22Q (Dutch), and A{beta} E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in A{beta} proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular A{beta} amyloidoses.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyloid {beta} (A{beta})1 peptide deposition in the walls of small arteries and less often in venules and capillaries of the cerebral cortex is a prominent feature of several neuropathological conditions characterized by dementia or stroke. These include Alzheimer's disease (AD), Down syndrome, and sporadic and autosomal dominant cerebral amyloid angiopathies (CAAs) (Ref. 1, and for a review, see Ref. 2). In sporadic AD, a large proportion of the cases show A{beta}-(1-40) and A{beta}-(1-42/43) accumulation in the media and adventitia of brain vessels. Although the contribution of such deposits to the pathophysiology of dementia is not known, their extent and localization are strongly influenced by the apoE genotype (3-5). Sporadic CAA accounts for ~5% of strokes, and in this condition, A{beta} wild type (wt) is the main component of amyloid deposits (Ref. 6, and for a review, see Ref. 7). The Flemish (A21G), Dutch (E22Q), and Italian (E22K) genetic variants of A{beta} show a remarkable tendency to accumulate in the leptomeningeal and cortical vasculature leading to fatal hemorrhagic strokes around the 5th decade of life (8-10). In addition, the Arctic (E22G) and Iowa (D23N) variants of A{beta} present clinically with dementia, and yet at the pathological level they also show prominent vascular deposits (11, 12). Interestingly the Dutch, Italian, and Iowa A{beta} variants, when aggregated, are capable of inducing in vitro toxicity upon endothelial and smooth muscle cells, suggesting a pathogenic relationship between A{beta} deposition, vascular pathology, and clinical manifestations (10, 12, 13). With regard to the underlying mechanisms that result in A{beta} accumulation in the vascular walls, amino acid substitutions clustered in the central region of A{beta} sequence such as E22Q, E22G, and D23N may facilitate a fast rate of aggregation, possibly due to a net charge effect at physiologic pH (11, 12, 14, 15). It has been postulated that such a property may be the key dominant gain of function in the pathogenesis of these diseases (16). In addition, a slower rate of reverse transport across the blood-brain barrier and perivascular drainage to the systemic circulation has been shown for the A{beta} Dutch variant as compared with A{beta} wt (17). The possibility of a defect in A{beta} degradation has recently gained experimental support as a mechanism contributing to its accumulation in the brain, particularly in the sporadic forms of A{beta}-associated diseases in which A{beta} wt is involved. Neprilysin (NEP), endothelin-converting enzymes (ECEs), IDE, angiotensin-converting enzyme, the plasmin system, and matrix metalloproteases among others are major proteases that may participate in the catabolic pathway of A{beta} in the brain (for a review, see Ref. 2). This process has been tested recently in vivo for NEP, IDE, and ECE using loss-of-function animal models (18-21). NEP is a membrane-bound zinc metalloprotease expressed in neurons and microvessels that shows an inverse correlation with the vulnerability to A{beta} deposition in the human brain (22, 23). Moreover the injection of a lentiviral vector expressing NEP in the central nervous system of transgenic mice carrying a pathogenic amyloid precursor protein (APP) human mutation resulted in a significant reduction in amyloid burden and neuronal damage (24). IDE is a 110-kDa zinc- and thiol-dependent peptidase present in the cytosol and peroxisomes and on the cell surface of a wide variety of cell types including neurons (25-27). Because of its Km for insulin in the nanomolar range, IDE is involved in the degradation of this hormone in vivo (28). Yet IDE is also capable of degrading many small peptides of unrelated sequence, several of which show amyloidogenic potential in vitro and in vivo such as glucagon, amylin, calcitonin, atrial natriuretic peptide, and A{beta}, including all the genetic variants of A{beta} associated with dementia or stroke (for reviews, see Refs. 29 and 30). Recently the accumulation of endogenous brain soluble A{beta} in mice lacking IDE expression after gene targeting has been shown (20, 21). This increase was gene dose-dependent for A{beta} species ending at positions 40 and 42, respectively, and strongly favors that IDE is a major physiologic A{beta}-degrading protease in the mammalian brain. The importance of NEP and IDE in the removal of A{beta} has been reinforced by the finding that transgenic overexpression of these proteases in neurons reduced brain soluble A{beta} levels and delayed amyloid plaque formation in APP transgenic mice (31). Moreover IDE activity and mRNA levels have been found to be lower in AD patients as compared with age-matched controls, and such a reduction may be influenced by the {epsilon} 4 allele of apoE (32, 33).

Despite the recent advances in our knowledge about A{beta} proteolysis by IDE in vitro and in murine models, little is known about the cellular and regional expression and activity of IDE in the brain and its possible role in A{beta}-related diseases. The aim of the present study was to investigate IDE in cortical microvessels isolated from human brain, a major target of A{beta} accumulation in AD and CAA. We examined its cellular localization and activity against insulin, synthetic A{beta}-(1-40) wt, and three major vasculotropic (Flemish, Dutch, and Italian) variants associated with hereditary stroke. To explore a possible pathogenic role of the protease, we determined the levels and activity of IDE in microvessels from late onset sporadic AD patients with CAA and non-demented age-matched controls.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic Peptides—Synthetic A{beta}-(1-40) peptides, A{beta} wt, A{beta} A21G, A{beta} E22Q, and A{beta} E22K, were synthesized by the W. M. Keck Foundation (Yale University, New Haven, CT). All the peptides were purified by reverse-phase high pressure liquid chromatography, and their purity was assessed by amino acid sequence analysis and laser desorption mass spectrometry. Lyophilized peptides were dissolved in deionized distilled water, centrifuged at 10,000 rpm for 30 min to eliminate large aggregates, and stored in aliquots at -85 °C. The state of aggregation of the A{beta} species used in this study was assessed by SDS-PAGE and Western blot as reported previously (30).

Expression and Purification of Recombinant Rat IDE—The plasmid pECE-IDE containing the coding region of rat IDE cDNA was kindly provided by Richard Roth, Stanford University. pECE-IDE was used as a template for the PCR amplification of a truncated IDE using the following primers: sense, 5'-AGCACAGGATCCATGAATAATCCGGCCAT-3' (nucleotides 139-155); antisense, 5'-TTCTCGAGGAGTTTTGCCGCCATGA-3' (nucleotides 3072-3056). Sites for BamHI and XhoI, respectively, were introduced to facilitate cloning. The PCR product was digested with BamHI and XhoI and cloned into pET-30a(+) from Novagen (Darmstadt, Germany) digested previously with the same restriction enzymes to generate the pET-IDE construct. Recombinant IDE-(42-1019) (rIDE) was expressed in E. coli BL21 and purified using a Hi Trap nickel-chelating column (Amersham Biosciences). The purity and activity of rIDE against 125I-insulin have been characterized previously (30).

Human Tissue—Brain tissue from two individuals (28 and 45 years old, respectively, with a postmortem delay of less than 6 h) who died of non-neurological causes were obtained from the Department of Pathology, Hospital Santojanni, Buenos Aires, Argentina. Samples were provided with a numeric code to preserve identity under a protocol approved by the Institutional Ethical Committee. Brain tissue samples from AD and non-demented (ND) cases were obtained from participants enrolled in the Brain Donation Program of Sun Health Research Institute (Sun City, AZ). Twelve autopsy cases were studied, including six sporadic late onset AD with CAA and six ND controls. Cases were defined as AD if they met the criteria established by the Consortium to establish a registry for Alzheimer's disease (CERAD) for definite or probable AD. Pathological staging was done following criteria established by Braak and Braak (34). The presence and severity of CAA was determined by thioflavine S staining and immunohistochemistry with anti-A{beta} (see below). Clinical and pathological data of both groups are summarized in Table I.


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  		TABLE I
Clinical and pathological features of AD cases with CAA and age-matched ND controls

 
Brain Microvessel Isolation—Brain cortical microvessels were isolated following the method described by Pardridge et al. (35) with minor modifications. Cortical tissue from frontal or occipital lobes was carefully dissected from leptomeninges and homogenized in 5 volumes of buffer A (15 mM HEPES, pH 7.4, 103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KHPO4, 25 mM NaHCO3, 10 mM glucose, 10 IU/ml heparin). An equal volume of 26% dextran (molecular weight, 65,000-85,000) in the same buffer was added, and the homogenate was centrifuged at 5,800 x g for 15 min at 4 °C. The pellet was resuspended in 10 volumes of buffer A and passed sequentially through 350-, 150-, and 70-µm nylon meshes under vacuum. The material that remained on top of the 150- and 70-µm meshes was recovered by inverting the filters and washing them with a stream of ice-cold buffer A. These fractions were pelleted at 1,000 x g for 10 min, resuspended to 1-2 ml in buffer A, and loaded on top of a preformed Percoll gradient (50% in buffer A, generated at 27,000 x g for 1 h). After centrifugation for 1,000 x g for 10 min, the middle white layer was collected, and the centrifugation on the Percoll gradient was repeated one more time. Fractions were examined under light microscopy and by SDS-PAGE and Western blot. A sample of brain cortical tissue was homogenized in buffer A and centrifuged at 1,500 x g for 5 min, and the supernatant was centrifuged at 100,000 x g for 1 h at 4 °C. This material was referred to as brain soluble fraction. The same procedure was followed to obtain a soluble fraction from rat liver as a source of IDE that has been previously purified using monoclonal 9B12 (see below) and characterized by SDS-PAGE (36).

Antibodies—Rabbit polyclonal BC2 antibodies were generated against a glutathione S-transferase fusion protein containing the sequence 97-273 of rat IDE as described previously (30) and affinity-purified using rIDE immobilized on CnBr-activated Sepharose 4B (Amersham Biosciences). Anti-IDE monoclonal antibodies 1C1 and 3A2 were obtained by immunization of BALB/c mice with rIDE. Both antibodies were characterized by ELISA and immunoprecipitation (see below), identified as IgG1, and purified from ascitic fluid following standard procedures. Anti-IDE monoclonal 9B12 antibody was a generous gift of Richard Roth (Stanford University). Monoclonal anti-CD31 was from DAKO (Carpenteria, CA); monoclonal anti-laminin and monoclonal anti-{alpha}-actin were from Sigma. Rabbit polyclonal anti-neuronal specific enolase (NSE) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-A{beta} 6E10 was obtained from Signet (Dedham, MA), and 3D6 was a gift from Elan Pharmaceuticals. Monoclonal mouse IgG1 against the papilloma virus E2 protein was kindly provided by Fernando Goldbaum, Fundación Instituto Leloir, Buenos Aires, Argentina.

Immunohistochemistry and Immunofluorescence—Brain tissue was fixed in 4% paraformaldehyde, and 40-µm-thick free floating sections were used for immunohistochemistry with anti-CD31 to label endothelial cells and 3D6 anti-A{beta} as described previously (37). Isolated microvessels where whole mounted on glass slides, fixed with methanol at -20 °C for 5 min, and blocked with 5% normal goat serum in PBS, pH 7.4, containing 0.05% Tween 20 (PBS-T) for 2 h at room temperature. Monoclonal 9B12 was incubated at 0.15 µg/µl, and monoclonal 1C1 was incubated at 0.25 µg/µl in blocking buffer overnight at 4 °C. Negative controls included a monoclonal IgG1 against E2 (see above) and omission of the first antibodies. For double immunostaining, 1C1 was co-incubated with a biotinylated endothelial cell marker Ulex europaeus agglutinin type 1 (Vector Laboratories, Burlingame, CA); biotin-labeled 1C1 was used in combination with anti-laminin and anti-{alpha}-actin. Slides were then washed with PBS-T and incubated for 1 h with Cy2-conjugated goat anti-mouse IgG (Amersham Biosciences) alone or with avidin-Alexa 568 (Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst 33342 at 5 µg/ml in PBS for 15 min at room temperature. Slides were visualized with a fluorescence microscope (Olympus BX50), and the captured images were processed with Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD). Confocal fluorescence microscopy was performed with a dual laser Olympus FV300 microscope, and images were analyzed with acquisition FluoView software, Version 3.3.

SDS-PAGE and Western Blots—Recombinant IDE; synthetic A{beta} peptides; proteins from microvessel homogenates, brain, and liver soluble fractions; and human erythrocytes at the indicated amounts were analyzed by 7.5 or 12.5% SDS-PAGE in Tris-Tricine gels. For Western blot, proteins were transferred to polyvinylidene difluoride membranes (Amersham Biosciences) and incubated with anti-IDE BC2 at 1:2,000, anti-{alpha}-actin at 1:500, and anti-NSE at 1:2,000. Immunoreactivity was detected with anti-rabbit or anti-mouse horseradish peroxidase-labeled IgG and enhanced chemiluminescence (ECL Plus, Amersham Biosciences). Immunoblots were scanned with Storm 840 and analyzed with ImageQuant 5.1 software (Amersham Biosciences).

Immunoprecipitation and IDE Activity Assay—To assess the presence and activity of endogenous IDE, isolated brain microvessels were sonicated (Branson 250 sonicator, output 4, three times, 10 s each time, on ice) in 10 volumes of PBS, pH 7.5, containing 2 mM phenylmethanesulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 10 IU/ml heparin (buffer B) and centrifuged at 5,000 x g for 10 min at 4 °C. After sonication, the complete disruption of microvessels was assessed by light microscopy. The supernatant was incubated with 7.5 µg/ml monoclonal anti-IDE 9B12 or monoclonal anti-{alpha}-actin and protein G-Sepharose for 2 h at room temperature. Immunocomplexes were washed five times in ice-cold buffer containing 0.4 M NaCl, 0.1 M phosphate, pH 7, without protein inhibitors. The protein G-Sepharose beads were then incubated in 50 µl of buffer consisting of 0.1 M phosphate, pH 7, containing 65,000 cpm of 125I-insulin (specific activity, 300 µCi/µg; kindly provided by Edgardo Poskus, University of Buenos Aires) for 1 or 2 h at 37 °C with constant agitation in the presence or absence of the following protease inhibitors at the indicated concentrations: EDTA (5 mM), 1,10-phenanthroline (1 mM), phenylmethanesulfonyl fluoride (2 mM), leupeptin (2 µg/ml), pepstatin A (2 µg/ml), aprotinin (2 µg/ml), thiorphan (50 µM), and phosphoramidon (50 µM). After incubation, beads were centrifuged at 1,500 x g for 5 min. Twenty microliters of the supernatant were removed, mixed with an equal volume of 8% SDS sample buffer, boiled, and resolved by 15% Tris-Tricine SDS-PAGE under non-reducing conditions. The amount of intact remaining 125I-insulin was estimated with a Storm 840 PhosphorImager (Amersham Biosciences), and proteolysis was expressed as the percentage of degradation determined by (A - B)/A x 100 where A represents the intensity of intact 125I-insulin incubated with protein G-Sepharose alone and B represents the intensity of intact 125I-insulin after incubation with 9B12 or anti-{alpha}-actin immunoprecipitates. As a positive control, 10-50 ng of purified rIDE were incubated with radiolabeled insulin in the presence of protein G-Sepharose, and degradation was assessed as described above. Within this range, there was a linear correlation between the amount of rIDE and the extent of 125I-insulin degradation (not shown).

A{beta} Proteolysis and Mass Spectrometry—Molecular masses of intact A{beta} peptides and the products generated by endogenous IDE were analyzed by mass spectrometry. Synthetic A{beta} peptides were incubated with 9B12 or anti-{alpha}-actin immunoprecipitates at 2 µM in 0.1 M phosphate buffer, pH 7, in the presence or absence of the indicated inhibitors for 1 h at 37 °C with constant agitation. After centrifugation at 1,500 x g for 3 min, 10-µl samples collected from the supernatants were passed through a reverse-phase ZipTip (Millipore, Billerica, MA) following the manufacturer's instructions. Samples were then analyzed on a Micro-mass TofSpec-2E (MALDI-TOF) mass spectrometer in linear mode using standard instrument settings at the New York University Protein Analysis Facility. Internal and/or external calibration was carried out using angiotensin I (average mass, 1296.5 Da) and insulin (average mass, 5733.5 Da). The profiles shown are representative of three independent experiments. To assess the extent of aggregation, aliquots of A{beta} peptides incubated with or without IDE were analyzed by SDS-PAGE and Western blot with 6E10 as described above.

IDE Quantification with Sandwich ELISA—Monoclonal antibody 3A2 was coated on microtiter plates (Nunc) at 3 µg/well in PBS, pH 7.4, for 16 h at 4 °C. After blocking with 3% bovine serum albumin in PBS for 4 h at room temperature, 20-100 µg of proteins from microvessels homogenates, as determined with a BCA kit (Pierce), were incubated in binding buffer (20 mM sodium phosphate, pH 7.5, 0.4 M NaCl containing 0.3% bovine serum albumin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 2 mM phenylmethanesulfonyl fluoride, 2 mM EDTA) for 16 h at 4 °C. Wells were washed with PBS containing 0.2% Tween 20, and biotinylated monoclonal 1C1 was incubated at 5 µg/ml in binding buffer for 2 h at room temperature. Wells were washed as above and incubated with peroxidase-conjugated streptavidin (Jackson Immunoresearch Laboratories, West Grove, PA) at 1:1,000 for 2 h at room temperature. Reactivity was developed with o-phenylenediamine and H2O2 for 10 min and stopped with 1 N SO4H2, and the absorbance was measured at 490 nm with a microtiter plate reader 550 (Bio-Rad). A calibration curve was established using rIDE as a standard. Nonspecific binding was determined using an unrelated mouse IgG1 as mock capture antibody and subtracted from experimental values. Levels of endogenous IDE were expressed as ng/mg of tissue protein. IDE concentrations in microvessels homogenates from ND and AD patients with CAA were determined in duplicate samples from two independent experiments and analyzed by Student's t test using GraphPad 3.0 software.

A{beta} Measurements in Isolated Microvessels—Microvessels were sonicated and centrifuged as described above to obtain a water-soluble supernatant. The pellets were resuspended in 10 volumes of 65% formic acid, sonicated for 1 min on ice, and centrifuged at 12,000 x g for 10 min at 4 °C. The formic acid-soluble supernatant was mixed with 10 volumes of 0.5 M sodium phosphate, 1 M Tris-HCl to neutralize the pH. Appropriate dilutions were used to determine the concentrations of A{beta}-(1-40) and A{beta}-(1-42) with a sandwich ELISA following manufacturer's instructions (Signet); concentrations were expressed as ng of A{beta}/mg of total protein. Determinations were done in duplicate in two independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IDE Localization in Human Brain Microvessels—Microscopic examination of the middle layer obtained from the Percoll gradient showed that it consisted mainly of long, branched, 20-30-µm-wide arterioles together with numerous capillaries and scattered clusters of astrocytic processes. To a much lesser extent, neuronal cell bodies were found, and no substantial contamination with erythrocytes was seen. The fraction obtained from the 350-µm mesh filtrate that remained on top of the 70-µm nylon mesh was highly enriched in vessels, and therefore this material was chosen for further analysis (Fig. 1, a and b). Immunofluorescence of whole mount microvessels with anti-IDE monoclonal antibodies showed a continuous staining along the walls of arterioles and capillaries with 9B12 together with a strong perinuclear staining that was more evident with 1C1 (Fig. 1, c and d). Confocal microscopy analysis after double labeling did not show co-localization of anti-IDE 1C1 reactivity with the endothelial cell surface marker U. europaeus agglutinin type 1 or with the basal lamina component laminin (Fig. 1, e and f). Yet 1C1 staining co-localized with {alpha}-actin reactivity within pericytes (Fig. 1, g-i) and smooth muscle cells (not shown). No signal above background was observed when the unrelated mouse IgG1 anti-E2 was used instead of 9B12 and 1C1 or when the first antibody was omitted (not shown). Together these results indicate that IDE was mainly localized within the cytoplasm of three major components of the microvascular wall: endothelial cells, pericytes, and smooth muscle cells.



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  		FIG. 1.
a, light microscopy examination of isolated microvessels from human brain cortical tissue. b, double labeling of microvessels with the endothelial cell surface marker U. europaeus agglutinin type 1 (red) and the basal lamina component laminin (green). c and d, immunofluorescence of brain capillaries with anti-IDE monoclonal antibodies 1C1 (c) and 9B12 (d). Arrowheads indicate perinuclear staining of pericytes and endothelial cells. Arrows indicate the homogenous staining of the vascular wall. e, double confocal immunofluorescence of brain capillary with U. europaeus agglutinin type 1 (red) and 1C1 (green). The arrowhead marks a pericyte, and the arrow indicates the nucleus of an endothelial cell. f, double confocal immunofluorescence with laminin (green) and biotinylated 1C1 (red). Arrowheads show peripheral basal lamina staining that does not colocalize with anti-IDE within the pericyte. g-i, colocalization of IDE detected by biotinylated 1C1 (red) with {alpha}-actin (green) in a capillary pericyte. Bars represent the following lengths: a, 40 µm; b, 50 µm; c, 35 µm; d, 25 µm; e, 15 µm; f, 10 µm; g-i, 50 µm.

 
IDE Activity in Isolated Microvessels—SDS-PAGE analysis of a microvessel homogenate followed by Coomassie Blue staining showed a pattern consistent with previous reports (35). More importantly, no hemoglobin was detected, indicating a negligible contamination with erythrocytes that are a known source of IDE (Fig. 2A). Western blot showed the expected strong immunoreactivity with anti-{alpha}-actin, a smooth muscle cell and pericyte protein, and a faint band that was positive with anti-NSE, while the opposite pattern was obtained from an aqueous 100,000 x g soluble fraction from human brain, further confirming the enrichment in vascular components of our preparation (Fig. 2B). When 10 µg of microvessels proteins were loaded on the gel, a 115-kDa protein that co-migrated with a band in the soluble fraction from human cerebral cortex and rat liver was detected by affinity-purified BC2 on Western blot. This 115-kDa component from the three preparations disappeared after adsorption with rIDE, indicating specificity of the reaction and its identity as endogenous IDE (Fig. 2C). Direct analysis by Western blot with 9B12 did not show the expected band of 115 kDa likely due to the low amount of IDE present in the sample. Therefore, the low speed soluble fraction obtained from microvessels after sonication was immunoprecipitated with 9B12 followed by Western blot detection with BC2. In this case, a robust 115-kDa protein that was not present after immunoprecipitation with the unrelated mouse anti-E2 was seen, consistent again with the endogenous IDE detected by direct BC2 Western blot (Fig. 2D) and with the immunofluorescence results. The {alpha}-actin and NSE patterns together with a similar intensity in the IDE signal after loading equal amounts of proteins from microvessels and brain soluble fractions strongly indicated a vascular origin of the protease.



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  		FIG. 2.
SDS-PAGE and Western blots of isolated cerebral cortical microvessels. A, Coomassie Blue-stained proteins. Lane 1, 30 µg of proteins from microvessels homogenate; lane 2, 2.5 µg of human hemoglobin. B, Western blot with anti-NSE (top) and anti-{alpha}-actin ({alpha}-act) (bottom). Lane 1, microvessels; lane 2, brain soluble fraction. Five micrograms of total proteins were loaded in each lane. C, Western blot with BC2. Lanes 1 and 4, microvessels homogenate; lanes 2 and 5, human brain soluble fraction; lanes 3 and 6, rat liver soluble fraction. Ten micrograms of proteins were loaded in each lane. The panel on the right shows the disappearance of IDE immunoreactivity after adsorption (ads.) of BC2 with 10 µg of recombinant rat IDE. D, immunoprecipitation of IDE from microvessels. Lane 1, immunoprecipitation with an unrelated mouse IgG1; lane 2, with 9B12. Western blot was developed with affinity-purified BC2 IgG (top). Fast green staining of IgG heavy chains from the same blot is shown (bottom). Left, molecular mass markers in kilodaltons. Each panel is representative of at least three independent experiments.

 
To assess the activity of endogenous IDE, 0.3 mg of microvessels proteins were subjected to immunoprecipitation with anti-IDE 9B12, the protein G-Sepharose-9B12 complexes were then incubated with 125I-insulin, and degradation was analyzed by SDS-PAGE followed by PhosphorImager quantitation. After 1 h at 37 °C, a degradation of 73.9 ± 4.8% of 125I-insulin by anti-IDE 9B12 immunoprecipitates (ipp) was observed. As a positive control, an almost complete proteolysis (98.7 ± 2%) was obtained in the presence of 50 ng of purified rIDE and protein G-Sepharose (Fig. 3). A strong inhibition in the anti-IDE 9B12 ipp was obtained with EDTA and 1,10-phenanthroline with a remaining 125I-insulin degradation of 16.5 ± 4%. Moreover 125I-insulin proteolysis was significantly lower (27.8 ± 3.1%) when mouse anti-{alpha}-actin IgG was used instead of 9B12 as a negative control (Fig. 3). As expected, no significant inhibitory effect upon 125I-insulin degradation was observed in 9B12 ipp in the presence of leupeptin, pepstatin A, aprotinin, phenylmethanesulfonyl fluoride, thiorphan, or phosphoramidon (not shown).



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  		FIG. 3.
Activity of endogenous IDE isolated from brain microvessels. A, SDS-PAGE and PhosphorImager analysis of 125I-insulin degradation by microvessel proteins immunoprecipitated with anti-IDE 9B12 or anti-{alpha}-actin, respectively, in the presence or absence of EDTA and 1,10-phenanthroline (Phen.) as indicated. B, densitometric quantitation of the endogenous IDE activity as described in A. Bars represent the mean ± S.E. of four independent experiments performed in duplicate.

 
Endogenous IDE Cleaves the Dutch, Flemish, and Italian A{beta} Variants—Since the sites of cleavage of IDE upon A{beta} have been extensively characterized (30, 38, 39), the activity of microvessel-derived IDE in 9B12 ipp upon synthetic A{beta}-(1-40) wt and genetic isoforms was then studied by mass spectrometry. A{beta}-(1-40) wt yielded fragments consistent with A{beta} peptides 1-13, 1-14, 1-18, 1-19, 1-20/4-23, 1-28, 15-40, and 20-40 (Fig. 4A). These peptides were in full agreement with the reported major sites of A{beta} cleavage by recombinant rat and human IDE and by IDE from brain soluble fractions. Moreover mass spectrometry analysis of A{beta}-(1-40) wt incubated with anti-{alpha}-actin ipp did not show the generation of peptides consistent with IDE activity with the exception of 1-20 and 21-40 (Fig. 4A), further supporting specificity. The Flemish variant A{beta} A21G was extensively cleaved, yielding nine major peptides that corresponded to positions 1-13, 1-14, 1-18, 1-19, 1-28, 4-13, 4-14, 15-40, and 20-40 (Fig. 4B), all consistent with IDE specificity. After incubation with 9B12 ipp, the Dutch variant A{beta} E22Q presented two major fragments corresponding to A{beta} peptides 1-19 and 1-20, an identical finding to what has been reported for the degradation of native A{beta} E22Q purified from HCHWA-D leptomeninges by rIDE (30) (Fig. 5, top panel). Such limited proteolysis of A{beta} E22Q correlated with a higher degree of oligomerization of the peptide on SDS-PAGE and Western blot with anti-A{beta} 6E10 as compared with the rest of the A{beta} peptides studied (not shown). The Italian variant A{beta} E22K yielded fragments 1-13, 1-14, 1-19, 1-20, 4-13, 4-14, and 4-19/5-20 (Fig. 6, top panel). None of the fragments obtained from these A{beta} genetic variants were seen after incubation with 9B12 ipp in the presence of EDTA and 1,10-phenanthroline (Figs. 5 and 6, bottom panels). Importantly neither thiorphan nor phosphoramidon inhibited degradation (not shown), further supporting that the proteolysis observed was due to endogenous IDE and not to other A{beta}-degrading metallopeptidases such as NEP or ECEs that may be present in brain cortical microvessels (23).



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  		FIG. 4.
A, mass spectrometry analysis of A{beta} wt degradation from brain microvessel proteins immunoprecipitated with anti-IDE 9B12 (top) or with anti-{alpha}-actin as a negative control (bottom). B, endogenous IDE cleavage products as in A from A{beta} A21G (Flemish variant). The insets show the calculated (Calc.) and observed (Obs.) masses and predicted fragments of A{beta} wt and A{beta}-(1-40) Flemish.

 



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  		FIG. 5.
Mass spectrometry of the A{beta}-(1-40) Dutch (E22Q) degradation products by endogenous IDE. A{beta} Dutch was incubated at 2 µM for 1 h at 37 °C with anti-IDE 9B12 ipp from microvessel proteins in the absence (top) or presence of 5 mM EDTA and 1 mM 1,10-phenanthroline (bottom). Insets show the observed (Obs.) and calculated (Calc.) masses of the A{beta} fragments.

 



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  		FIG. 6.
Mass spectrometry of the A{beta}-(1-40) Italian (E22K) variant after degradation with IDE. A{beta} E22K was incubated with microvessel proteins immunoprecipitated with anti-IDE 9B12 in the absence (top) or presence of 5 mM EDTA and 1 mM 1,10-phenanthroline (bottom). Insets show the observed (Obs.) and calculated (Calc.) masses of A{beta} species.

 
A{beta} and IDE Levels in Microvessels from AD Cases with CAA—To explore a possible role of IDE in the pathogenic mechanisms of cerebrovascular A{beta} amyloidosis, cortical microvessels were isolated from six AD cases with CAA and six age-matched ND controls whose clinical and pathological features are summarized in Table I. Immunostaining of brain tissue sections with antibody 3D6, which recognizes the amino terminus of A{beta}, detected a profuse vascular deposition of the peptide in AD-CAA (Fig. 7, A and B). The yield of microvessels was similar in both groups as assessed by light microscopy and protein content of the water-soluble fraction after sonication (0.49 ± 0.1 and 0.44 ± 0.08 mg/ml for ND controls and ADCAA, respectively). The presence and abundance of A{beta}-(1-40) and A{beta}-(1-42) in microvessels were further determined with sandwich ELISAs in the water-soluble and formic acid-soluble fractions. Water-soluble A{beta}-(1-40) was 2-fold higher in ADCAA as compared with ND controls (30.9 and 16.7 ng/mg, respectively), while formic acid-soluble peptide was very low in ND controls and strongly increased in AD-CAA (10.1 and 1030 ng/mg, respectively) (Fig. 7, C and D). A similar profile was obtained for A{beta}-(1-42) with water-soluble levels of 1.75 and 5.37 ng/mg in ND controls and AD-CAA, respectively. Formic acid-soluble A{beta}-(1-42) was also robustly elevated in AD-CAA (363 ng/mg) as compared with ND controls (57.5 ng/mg) (Fig. 7, C and D). To obtain an accurate quantification of IDE in brain microvessels, we developed a sandwich ELISA using two monoclonal antibodies, 3A2 and 1C1, raised against rIDE that were able to immunoprecipitate the endogenous protease from different tissues and species (Fig. 8A). Using 3A2 as capture antibody and biotinylated 1C1 as reporter, a specific and linear signal (r = 0.99) was obtained up to 0.2 µg/ml with rIDE as a standard (Fig. 8B). The water-soluble supernatants were then analyzed by sandwich ELISA with antibodies 3A2 and 1C1. The levels of IDE were significantly higher in AD cases with CAA (241.2 ± 19 ng/mg) as compared with the ND control group (167.1 ± 24 ng/mg), p = 0.04, determined by Student's t test (Fig. 9A). A possible contribution of IDE from red blood cells in the AD-CAA microvessels in which microhemorrhages are known to occur was negligible since the amount of IDE after sonication of a red cell pellet was 95 ± 9.6 ng/mg, and as mentioned above, no hemoglobin band was detected by SDS-PAGE. More importantly, the IDE concentrations obtained by ELISA reflected the presence of full-length 115-kDa IDE as shown by Western blots with BC2 followed by densitometry (Fig. 9B).



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  		FIG. 7.
Characterization of AD-CAA and ND microvessels by immunohistochemistry and sandwich ELISA of A{beta} A and B, double immunostaining of microvessels isolated from AD-CAA cases and ND controls, respectively, with anti-CD31 (purple) and anti-A{beta} 3D6 (brown). Arrows indicate vascular amyloid deposits. Bars represent a length of 50 µm. C, levels of A{beta}-(1-40) (A{beta}40) and A{beta}-(1-42) (A{beta}42) peptides in water-soluble fractions. D, levels of A{beta} peptides solubilized in formic acid. Microvessels isolated from ND and AD-CAA cases were homogenized by sonication, and fractions were obtained and analyzed as described under "Experimental Procedures." Mean values ± S.E. from two independent experiments performed in duplicate are depicted.

 



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  		FIG. 8.
Sandwich ELISA used to determine IDE levels. A, immunoprecipitation of IDE with monoclonal antibodies 3A2 and 1C1 and visualized by Western blot with polyclonal anti-IDE BC2. Lanes 1, 3, and 5, rat liver; lanes 2, 4, and 6, human brain; lane 7, rIDE. ur IgG, immunoprecipitation with an unrelated monoclonal IgG1. On the left, molecular mass marker in kilodaltons. B, calibration curve of sandwich ELISA with rIDE using monoclonal 3A2 as capture antibody and biotinylated 1C1 as a reporter. Results represent the mean ± S.E. of three independent experiments performed in triplicate.

 



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  		FIG. 9.
Quantification of IDE in microvessels from AD patients with CAA and age-matched ND controls. A, sandwich ELISA of endogenous IDE in homogenates from AD-CAA ({blacksquare}) and control microvessels ({blacktriangleup}). Each point represents the mean of two independent experiments performed in duplicate; the horizontal bars indicate the mean of each group. **, p = 0.04 as determined by Student's t test. B, representative Western blots of individual samples from AD-CAA and ND controls. IDE levels, as determined by ELISA, were 163, 93.7, and 294 ng/mg for AD-CAA-1, ND, and AD-CAA-2, respectively. Upper set, 20 µg of proteins were loaded, and IDE was detected with BC2. Lower set, 5 µg of proteins were loaded, and the Western blot was developed with anti-{alpha}-actin ({alpha}-act) to show normalization. On the left, molecular mass markers in kilodaltons.

 
IDE Activity Is Reduced in Microvessels with CAA—To determine whether the higher IDE protein levels resulted in a higher activity, aliquots from the microvessel water-soluble fractions from ND controls and AD-CAA cases analyzed by ELISA were pooled and subjected to the 9B12 immunoprecipitation assay as characterized above using 125I-insulin as a substrate. The degraded fraction of radiolabeled insulin was significantly lower in the AD-CAA group (12.2 ± 6.3% at 1 h and 24.7 ± 4.3% at 2 h) when compared with the ND group (32.6 ± 3.1% at 1 h and 64.3 ± 4.5% at 2 h), p < 0.05, Student's t test (Fig. 10A). This extent of degradation was expected for the amount of IDE used for immunoprecipitation (~15 ng) in the microvessels from ND controls, while it was unexpectedly low in the AD-CAA samples. To resolve whether the reduced degradation of 125I-insulin in AD-CAA ipp was due to a lower efficiency in the immunoprecipitation of IDE or to the fragmentation of the protease, 9B12 ipp were analyzed by Western blot with polyclonal BC2. As shown in Fig. 9B, similar amounts of 115-kDa IDE with the same electrophoretic mobility were pulled down by 9B12-protein G-Sepharose from ND and ADCAA samples, ruling out such possibilities (Fig. 10B).



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  		FIG. 10.
Activity of IDE from microvessels isolated from ADCAA and ND brain samples. A, degradation of 125I-insulin by pooled samples of microvessel homogenates from patients with AD-CAA ({blacksquare}) and ND controls ({blacktriangleup}) as determined with the 9B12 immunoprecipitation assay. **, p < 0.05 as determined by Student's t test. Results represent the mean ± S.E. of two independent experiments performed in triplicate. B, upper set, Western blot with BC2 of 9B12 ipp from pooled samples from ND and AD-CAA microvessels. Middle set, Coomassie Blue staining of bovine serum albumin, used for stabilization of the tracer, to show normalization of the radiolabeled insulin input in the degradation assay. Lower set, representative PhosphorImager scan of 125I-insulin after incubation for 2 h at 37 °C with the 9B12 ipp from the pooled samples as indicated in the upper set.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that IDE is enzymatically active in cortical brain microvessels isolated from human brain. The pattern of immunofluorescence with specific antibodies 9B12 and 1C1 indicated that IDE was present mainly within the cytoplasm of pericytes, endothelial cells, and smooth muscle cells in arterioles and capillary walls where A{beta} accumulates heavily in AD and CAA. Immunoprecipitation and Western blots showed a single 115-kDa isoform of IDE in a soluble fraction obtained from microvessels that co-migrated with IDE from a high speed soluble fraction from human brain and rat liver. Together immunofluorescence data and Western blots after normalization of IDE reactivity with anti-{alpha}-actin and -NSE strongly favored that the sources of the protease in our microvessel preparations were cellular components of the vessel wall. Among these, we cannot exclude a contribution from astrocytic processes and nerve terminals that innervate vessels. Although IDE has been found in murine microglia (40), it seems that neurons are a major source of the protease in the cerebral cortex (27, 41). With the combination of 9B12 immunoprecipitation and MALDI-TOF mass spectrometry, we determined that endogenous IDE was capable of degrading A{beta}-(1-40) wt and three major vasculotropic peptides associated with human disease, namely the Flemish (A21G), Dutch (E22Q), and Italian (E22K) variants. The cleavage sites upon A{beta} peptides were fully consistent with previous reports by our and other groups (30, 38, 39, 42). The more restricted cleavage found for synthetic A{beta} Dutch in this study and for A{beta} Dutch isolated from HCHWA-D leptomeninges in a previous work (30) may reflect the faster aggregation of this variant as compared with A{beta} wt, Flemish, or Italian peptides (10, 14, 15). In such a case, Phe19-Phe20 and Phe20-Ala21 may be major initial sites of hydrolysis in the soluble monomeric peptide and, therefore, more easily detected. Importantly none of the A{beta} fragments generated by endogenous IDE were found to be toxic to primary cell cultures as reported for synthetic A{beta}-(1-40) wt and a recombinant IDE (38).

It is now accepted that the clearance of soluble A{beta} from the brain is a complex process composed of three major mechanisms: (i) transport to the systemic circulation along perivascular drainage pathways (43), (ii) receptor-mediated reverse transport across the blood-brain barrier (44), and (iii) intracellular and extracellular proteolytic degradation (for a review, see Ref. 2). The mechanisms by which A{beta} and its vasculotropic variants accumulate within the walls of small vessels in the cerebral cortex and leptomeninges are not known. Yet it is possible that a defect in clearance pathways may drive the steady-state levels of soluble A{beta} beyond a concentration barrier for peptide oligomerization. It has been shown that after oligomers are formed, A{beta} is highly resistant to degradation by physiologic and non-physiologic peptidases (30, 45). IDE has been found to be lower in AD brains as compared with age-matched controls (32, 33). In a first report from our group, insulin and A{beta} degradation by an IDE-like protease in a high speed supernatant from cerebral cortex correlated with lower levels of IDE on Western blot (32). A more recent study showed that IDE protein and mRNA were reduced in hippocampal samples from AD patients carrying the apoE {epsilon} 4 allele compared with AD without such allele and ND controls (33). Therefore, our result in the present study of higher IDE levels in AD-CAA cortical microvessels was rather unexpected. Yet several proteases related to A{beta} degradation have been shown to be up-regulated in AD brains, such as matrix metalloprotease-9 (46), cathepsin D (47, 48), and cathepsin S (49). The urokinaseand tissue-type plasminogen activators and matrix metalloprotease-9 are elevated in APP transgenic mice (50, 51). Furthermore cultured glial, neuronal, and cerebrovascular smooth muscle cells responded to A{beta} treatment with a robust overexpression of matrix metalloproteases and urokinase- and tissue-type plasminogen activators (50, 52-54). Studies in transgenic mice expressing human APP mutants with neuron-specific promoters show an age-dependent cerebrovascular A{beta} accumulation (55-57), and vascular A{beta} deposition in AD brains has been correlated with a slower rate of perivascular drainage of the peptide (58). It seems possible that a relentless exposure of microvessels to A{beta} in vivo promotes the chronic overexpression of IDE. If this is the case, in vitro experiments are needed to determine which vascular cell types may contribute to IDE overproduction under A{beta} stimulation. Despite the higher levels of IDE in AD-CAA microvessels, the extent of degradation of radiolabeled insulin in the anti-IDE 9B12 immunoprecipitation assay was strongly reduced as compared with ND controls. The concentration of water-soluble A{beta}-(1-40) in the AD-CAA homogenate, although higher than in normal microvessels, was ~1.5 nM. Due to the known Km for A{beta}, between 0.8 and 2 µM (32, 59), competitive inhibition by soluble A{beta} could not account for the reduced activity upon 125I-insulin found in our assays. Along with the up-regulation of proteases, several protease inhibitors appear to be increased in AD brains including {alpha}1-antichymotrypsin, cystatin C, the Kunitz-type domain of APP, and anti-thrombin III (60-63). Moreover, in APP transgenic mice, chronically elevated A{beta} in the brain is associated with the up-regulation of plasminogen activator inhibitor-1 and inhibition of the tissue-type plasminogen activator-plasmin system (64). It has been shown that IDE activity in the liver may be regulated by heat-resistant, low molecular weight endogenous inhibitors, although their complete characterization has been elusive (36, 65). In a recent report, one of such inhibitors has been proposed to be ubiquitin, which co-purified with IDE from mouse leukemic splenocytes and bound to it non-covalently in vitro (66). Although the presence of IDE inhibitors in the brain and its microvasculature has not been described, our results raise the possibility that in CAA microvessels IDE is inhibited or inactivated, leading to a defective A{beta} degradation that may be pathogenically relevant in dementia and stroke.


   FOOTNOTES
 
* This work was supported by a grant from the Alzheimer's Association, Agencia Nacional de Promoción Científica y Tecnológica Grant 05-10599, and National Institutes of Health Grants AG10491, AG08721, NS38777, and AG05891. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 54-11-4-964-8288; Fax: 54-11-4-962-5457; E-mail: edcast{at}ffyb.uba.ar.

1 The abbreviations used are: A{beta}, amyloid {beta}; AD, Alzheimer's disease; apoE, apolipoprotein E; APP, amyloid precursor protein; CAA, cerebral amyloid angiopathy; ECE, endothelin-converting enzyme; IDE, insulin-degrading enzyme; rIDE, recombinant IDE; ipp, immunoprecipitates; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NEP, neprilysin; NSE, neuronal specific enolase; PBS, phosphate-buffered saline; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl-]glycine; wt, wild type; ND, non-demented; ELISA, enzyme-linked immunosorbent assay. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas Beach of Sun Health Research Institute for the neuropathological diagnosis of AD and ND brains.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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