Amyloid- Peptide Levels in Brain Are Inversely Correlated with Insulysin Activity Levels in Vivo

Amyloid-␤ peptide levels in brain are inversely correlated with insulysin activity levels in vivo

Bonnie C. Miller*†‡, Elizabeth A. Eckman†§, Kumar Sambamurti†§¶, Nicole Dobbs*, K. Martin Chow ሻ, Christopher B. Eckman§, Louis B. Hershሻ**, and Dwain L. Thiele*,**

*Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX 75390-9151; §Mayo Clinic, Jacksonville, FL 32224; and ࿣Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0298

Communicated by Steven L. McKnight, University of Texas Southwestern Medical Center, Dallas, TX, March 14, 2003 (received for review January 22, 2003) Factors that elevate amyloid-␤ (A␤) peptide levels are associated in vitro (9), including ␤-endorphin (10). To gain insight into the with an increased risk for Alzheimer’s disease. Insulysin has been contribution of insulysin to A␤ degradation in vivo, we have identified as one of several proteases potentially involved in A␤ examined the steady-state levels of endogenous A␤ peptides in degradation based on its hydrolysis of A␤ peptides in vitro. In this a mouse model in which insulysin gene expression has been study, in vivo levels of brain A␤40 and A␤42 peptides were found disrupted by a gene-trap insertion. to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the Materials and Methods level of the ␥-secretase-generated C-terminal fragment of the A␤ Mice. Omnibank B6.129 mice containing a gene-trap vector precursor protein in the insulysin-deficient mouse also was found. VICTR (viral construct for trapping; ref. 11) inserted in intron In mice heterozygous for the insulysin gene trap, in which insulysin 1 of the gene encoding insulysin were obtained from Lexicon activity levels were decreased Ϸ50%, brain A␤ peptides were Genetics (The Woodlands, TX). The standard designation increased to levels intermediate between those in wild-type mice ‘‘IDE’’ that is used to refer to the insulysin-encoding gene is and homozygous insulysin gene-trap mice that had no detectable based on the original name for insulysin, insulin-degrading insulysin activity. These findings indicate that there is an inverse enzyme. Mice heterozygous for the gene trap were bred to correlation between in vivo insulysin activity levels and brain A␤ produce ‘‘knockouts’’ (IDEϪ/Ϫ) containing the gene trap in both peptide levels and suggest that modulation of insulysin activity alleles as well as littermate wild-type controls and heterozygotes. may alter the risk for Alzheimer’s disease. The insulysin-deficient mice are healthy and exhibit no overt phenotype in behavior or development, or upon gross examina- myloid-␤ (A␤) peptide-containing senile plaques are a tion during necropsy, for at least 10 weeks. Blood glucose and Ϫ/Ϫ Aprominent feature of the pathology of Alzheimer’s disease insulin levels in IDE mice determined4hafterrefeeding a (AD) and occur consistently in AD of all etiology, from early- standard diet to overnight-fasted animals are not different from onset, familial-linked AD to late-onset AD of indeterminate those in wild-type littermates, suggesting that insulin degrada- origin (1). A␤ is formed from the amyloid precursor protein tion and activity are not severely perturbed by the deficiency. (APP) by sequential enzymatic processing. A ␤-secretase cleav- Male offspring between 7 and 10 weeks of age were used unless age first yields the 99-aa C-terminal fragment (CTF) of APP, otherwise noted. The mouse genotypes were determined by PCR CTF␤, which then is cleaved by ␥-secretase to release A␤ amplification of DNA that was purified by standard techniques peptides, predominately A␤40 and A␤42, and the CTF␥ peptides from tail snips with primers to a portion of the VICTR gene-trap Ј of 49–51 residues (2). sequence (BTK18F, 5 -CCATGGCTCCGGTAGGTCCAG, Ј The proteolysis of APP to yield A␤ peptides is a normal and BTK84R, 5 -TAATGCAGGTAGCTCCCAGA; 1 min at physiologic process observed in multiple cell types, although the 94°C followed by 1 min at 60°C for 35 cycles) to distinguish ϩ/ϩ endogenous function of APP processing and its products is still wild-type (IDE ) littermates from those carrying either one or NEUROSCIENCE Ϫ/ϩ Ϫ/Ϫ not well defined (3). To date, all of the genetic mutations linked two copies of the gene-trap vector. IDE and IDE mice to AD result in increased A␤ accumulation, albeit by distinct were distinguished by determining VICTR gene-trap copy num- mechanisms. ber vs. IDE gene copy number by quantitative Southern slot blot ␮ Although considerable effort has been directed toward gen- of the purified DNA. Serial DNA dilutions (2.0, 1.0, and 0.5 g) erating specific inhibitors of the ␤- and ␥-secretases as a means from mice to be genotyped, as well as DNA standards from mice of preventing A␤ formation (4), the mechanism of A␤ clearance whose genomes contained either two or no copies of the gene also is of considerable interest because the steady-state concen- trap, were slotted onto duplicate nitrocellulose filters by using a trations of A␤ peptides are dependent on both their rates of Minifold Slot Blotter II (Schleicher & Schuell). The filters were synthesis and their rates of clearance. Recent studies suggest that incubated with hybridization probes to either the neomycin an important route of A␤ clearance is through hydrolytic cleav- region of the gene-trap vector (corresponding to nucleotides age by proteases and peptidases (recently reviewed in refs. 2996–3271 of pPGKneo-II, GenBank accession no. AF335420) 36–38). The peptidase insulysin (EC 3.4.24.56) is one of the or to IDE (corresponding to nucleotides 1256–1781 of the IDE enzymes that has been suggested as a candidate A␤-degrading cDNA sequence, GenBank accession no. BC041675). The enzyme primarily based on its ability to degrade A␤ peptides in probes were synthesized by PCR amplification of cloned se- vitro (5–7). Insulysin is a zinc metalloprotease, originally identified as an Abbreviations: AD, Alzheimer’s disease; A␤, amyloid-␤; APP, amyloid precursor protein; insulin-degrading enzyme (8), that migrates with reported mo- CTF, C-terminal fragment; DIG, digoxigenin; IDE, insulin-degrading enzyme. ͞ lecular masses of 110–115 kDa on SDS polyacrylamide gels and †B.C.M., E.A.E., and K.S. contributed equally to this work. has no demonstrated posttranslational modifications. The ob- ‡To whom correspondence should be addressed at: Department of Internal Medicine, served molecular mass of insulysin is consistent with the use of University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX the second of its two potential translation initiation sites, al- 75390-9151. E-mail: [email protected]. though N-terminal sequencing of authentic insulysin has not ¶Present address: Department of Neuroscience and Physiology and Center on Aging, been reported. A limited number of peptides in addition to Medical University of South Carolina, Charleston, SC 29425. insulin and A␤ also have been identified as insulysin substrates **L.B.H. and D.L.T. contributed equally to this work.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1031520100 PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 6221–6226 Downloaded by guest on October 3, 2021 quences, incorporating digoxigenin (DIG)-labeled nucleotides supernatant fractions were resolved by electrophoresis in 10% for immunodetection. After washing to remove unbound probe, Tris⅐HCl Ready gels (Bio-Rad), using standard sample and the blots were incubated sequentially with an alkaline phos- electrophoresis buffers containing SDS, and blotted to nitrocel- phatase-labeled anti-DIG antibody and the chemiluminescent lulose (SS Protran; Schleicher & Schuell) electrophoretically. substrate CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane- Immunodetection was performed by using the primary antibod- 3,2Ј-(5Ј-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate] ies indicated in the figure legends, horseradish peroxidase- and exposed to autoradiographic film. All reagents for preparing conjugated anti-rabbit Ig as the secondary antibody, and the DIG-labeled probes and for DIG detection were from Roche ECL (enhanced chemiluminescence) Western Blotting Analysis Molecular Biochemicals. Probe synthesis, hybridization, and System from Amersham Pharmacia. Benchmark prestained pro- detection were performed according to the manufacturer’s tein standards (GIBCO͞BRL) were used for molecular weight protocols. Densitometric quantitation of the scanned images was estimation. performed with IMAGEQUANT software (Molecular Dynamics). Confirmation of the genotype by semiquantitative RT-PCR Insulysin Activity Measurements. Insulysin activity in 10,000 ϫ g and͞or Western blot analysis to measure insulysin mRNA or supernatant fractions of tissue homogenates prepared in 10 mM protein expression in at least one tissue was performed for each potassium phosphate buffer, pH 7.4, containing 0.2 M sucrose mouse by using the techniques described below. was measured by the generation of ␥-endorphin from ␤-endorphin (10). Reaction mixtures of 100 ␮l containing 30 ␮M RT-PCR. Both standard and real-time RT-PCR techniques were ␤-endorphin and Ϸ20 ␮g of liver extract in 100 mM potassium used to examine insulysin mRNA levels. For both, total RNA phosphate buffer, pH 7.4, were incubated for 10 min at 37°C. The purified by acid͞chloroform͞phenol extraction (12) was reverse- reaction was stopped by the addition of 10 ␮l of 5% trifluoro- transcribed with Superscript II (GIBCO͞BRL) by using oli- acetic acid, and the reaction products then were separated by go(dT) for priming and following the manufacturer’s suggested reverse-phase HPLC on a Vydac C4 column (Hesperia, CA), protocol for RNA messages with a high content of guanine and using a linear gradient from 0.1% trifluoroacetic acid in 95% cytosine residues. Control samples (-RT) were treated identically water͞5% acetonitrile to 0.1% trifluoroacetic acid in 50% except that the reverse transcriptase enzyme was not added to water͞50% acetonitrile. Peptides were detected by their absor- the reactions. For semiquantitative comparison within the linear bance at 214 nm and quantified by peak area. phase of amplification, PCR with the intron-spanning IDE primers 12F, 5Ј-GGAAGCGTTCGCCGAGATCGCA, and ELISA Quantitation of Brain A␤ Peptides. Brains were removed Ј 614R, 5 -TCTGAATCGACAGCGTTCAC, was performed by immediately after euthanasia of the mice by CO2 narcosis, frozen using a program of 94°C, 55°C, and 72°C for 45 sec each over a in liquid nitrogen, and stored frozen until extraction. Brains were range of cycles with a technique we have described previously extracted in 0.2% diethylamine in 50 mM NaCl and centrifuged (13). Dimethyl sulfoxide (0.1%) was added to the amplification at 20,000 ϫ g for1hat4°C to remove insoluble material. 1 reactions to reduce DNA secondary structure. For detection, the Supernatant fractions were neutralized by the addition of ⁄10 PCR products were separated by agarose gel electrophoresis, volume of Tris⅐HCl, pH 6.8, and analyzed by sandwich ELISA for blotted to nylon membranes, and hybridized with a DIG-labeled A␤40 and A␤42 essentially as described previously (14, 15) by probe corresponding to nucleotides 12–614 of the IDE cDNA using the BNT77͞BA27 and BNT77͞BC05 antibody systems to sequence. PCR of actin from the same reverse-transcribed detect A␤40 and A␤42, respectively. We are grateful to Takeda samples was performed similarly over a range of cycles with DIG (Osaka) for their generous gifts of BNT77, BA27, and BC05. nucleotides incorporated during PCR for direct detection as we have described previously (13). PCR reagents were all from Western Blot Analysis of Brain APP and C-Terminal Fragments. Equal Perkin–Elmer. To determine relative insulysin mRNA levels by amounts of protein in neutralized diethylamine brain extracts real-time PCR with the comparative cycle threshold method, the were resolved by SDS͞PAGE on 10–20% Tris-Tricine peptide IDE-specific primers ex1͞2-F, 5Ј-CACCTTGCGCTCCATCCT, gels (Bio-Rad) and electroblotted. The O443 antibody against and ex1͞2-R, 5Ј-GCCGGATTACTCATTGTGCTGTA were the C-terminal 20 residues of APP (16) was used for immuno- used. The PCR products were detected in real time with an detection of APP, CTF␤, and CTF␥ by using the chemilumi- IDE-specific Taqman minor groove binder probe, 5Ј- nescent SuperSignal Western blotting system from Pierce. The TGGGAATCCACACAGTC. Insulysin mRNA levels were nor- relative levels of APP and CTF␥ were determined by densitom- malized to 18S rRNA levels that were determined in the same etry of the scanned images by using IMAGEQUANT software reverse-transcribed samples by using a commercially available (Molecular Dynamics). 18S rRNA TaqMan assay system (Applied Biosystems) that specifically detects 18S cDNA and not genomic sequences. Statistical Methods. The A␤40 and A␤42 peptide determinations were analyzed by employing a two-way ANOVA, with IDE allele Western Blot Analysis of Insulysin. Two different antibodies to type and age group as the two factors. The post hoc Student– insulysin were elicited by standard techniques in rabbits. The Newman–Keuls test, with a type I error of 0.05, was applied to rIDE4020 antibody was elicited by immunization with nickel allele type to further assess statistically significant results from agarose-purified recombinant rat insulysin with an N-terminal the ANOVA. The testing was implemented in the SAS (Cary, six-histidine tag, which was expressed by a baculovirus system NC) statistical software package. (PharMingen) in Sf9 insect cells. The hIDE7–24 antibody was elicited by immunization with a synthetic peptide that corre- Results sponds to amino acids 7–24 (relative to the second ATG) of To investigate the contribution of insulysin to A␤ degradation in human insulysin that was coupled to keyhole limpet hemocyanin vivo, we examined the steady-state level of endogenous A␤ by a C-terminal cysteine residue (Pierce). Tissue homogenates peptides in mice deficient in insulysin expression. A mouse were prepared in the 10 mM Tris⅐HCl, pH 7.8, lysis buffer that model in which the IDE gene was disrupted by insertion of a we have described previously (13), which contains protease VICTR gene trap into intron 1 was used for these studies. To inhibitors but no detergent, and centrifuged for 10 min at confirm that insulysin expression was eliminated by this disrup- 10,000 ϫ g to remove debris. Protein content of the resulting tion, we initially examined insulysin mRNA and immunoreactive supernatant fractions was determined by the bicinchoninic acid protein levels. This question was of particular importance be- method (Sigma), using BSA as the standard. Proteins in the cause intron 1 is upstream of the second of two potential protein

6222 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1031520100 Miller et al. Downloaded by guest on October 3, 2021 Fig. 2. Liver insulysin activity in IDE gene-trap mice. Shown are HPLC chromatograms of assays of insulysin activity in liver extracts from an IDEϩ/ϩ mouse (trace B), an IDEϩ/Ϫ mouse (trace C), and an IDEϪ/Ϫ mouse (trace D), as well as a control incubation with the substrate ␤-endorphin (␤-Ep) alone (trace A). The chromatograms are offset so that all peaks can be seen. Peak a, ␤-Ep-(17–31); peak b, ␤-Ep-(18–31); peak c, ␤-Ep-(1–17) (␥-endorphin), peak d, ␤-Ep-(1–18); peak e, an unidentiﬁed secondary cleavage product.

␤ ␥ Fig. 1. Liver insulysin expression in IDE gene-trap mice. (A) Equal amounts of -endorphin to -endorphin (10). Insulin was not used as a total RNA from sex-matched littermates carrying one (IDEϪ/ϩ), no (IDEϪ/Ϫ), or substrate because insulysin is only one of several enzymes that two (IDEϩ/ϩ) copies of the wild-type IDE allele were reverse-transcribed (ϩRT) can degrade insulin, whereas insulysin is the only enzyme known or treated identically but without addition of reverse transcriptase (ϪRT). to metabolize ␤-endorphin to ␥-endorphin. As shown in Fig. 2, Portions of the same reverse-transcribed RNA were subjected to PCR with no activity was detected in the IDEϪ/Ϫ mice, whereas hetero- insulysin-speciﬁc primers (Top) and ␤-actin-speciﬁc primers (Middle) over a Ϸ range of cycles (22, 24, and 26 cycles for the ϩRT insulysin samples and 26 cycles zygote animals expressed 50% of the activity observed in

for the ϪRT insulysin samples; 12, 14, and 16 for the ϩRT actin and 16 cycles wild-type mice. In brain, insulysin mRNA levels (Fig. 3A) and NEUROSCIENCE ϩ/Ϫ for the ϪRT actin sample). The insulysin PCR products were displayed by immunoreactive protein (Fig. 3B) in heterozygote IDE mice hybridization with a DIG nucleotide-labeled insulysin probe and chemilumi- also were approximately half of those detected in wild-type nescent detection as described in Materials and Methods. Densitometric littermate controls, and the ␥-endorphin-generating activity was quantitation of the insulysin products is displayed in Bottom.(B) Equal 56 Ϯ 9% of wild type (n ϭ 6 determined from three independent ␮ ͞ amounts of liver protein (10 g per lane) were resolved by SDS PAGE and sets of mice consisting of two IDEϪ/ϩ and one IDEϩ/ϩ sex- analyzed by Western blotting with an antibody elicited with recombinant rat insulysin. The location of the 113-kDa insulysin protein is indicated. matched littermates). No insulysin protein (Fig. 3C) or mRNA (not shown) is detected in brain from IDEϪ/Ϫ mice. After characterization of insulysin mRNA, protein, and ac- translation initiation sites in the IDE gene (17, 18). In mice tivity levels in the insulysin gene-trap mice, we examined the containing two copies of the gene trap (IDEϪ/Ϫ), no insulysin effect of absent or diminished insulysin on A␤ accumulation in mRNA was detected in liver, a tissue with a normally high level the brain. Both A␤40 (Fig. 4A) and A␤42 (Fig. 4B) were Ϫ Ϫ of insulysin expression, whereas an intermediate decrease was increased, 1.6- and 1.4-fold, respectively, in IDE / mice com- found in heterozygote (IDEϩ/Ϫ) mice (Fig. 1A). An intermediate pared with wild-type littermate controls, with means and SDs for decrease in immunoreactive insulysin protein also was found in the combined age groups of 1.81 Ϯ 0.17 (n ϭ 7) compared with liver from the heterozygote mice, whereas no insulysin protein 1.17 Ϯ 0.16 (n ϭ 5) pmol͞g, respectively, for A␤40 and 0.57 Ϯ was detected in the IDEϪ/Ϫ mice (Fig. 1B). In addition to the 0.03 (n ϭ 7) compared with 0.42 Ϯ 0.04 (n ϭ 5) pmol͞g, 113-kDa insulysin protein, the insulysin antibodies also detect a respectively, for A␤42. Intermediate increases were observed in Ϫ ϩ prominent protein band of Ϸ120 kDa and a less prominent heterozygous IDE / mice; A␤40 was 1.57 Ϯ 0.17 pmol͞g and doublet of Ϸ75 kDa in some tissues in which insulysin is A␤42 was 0.51 Ϯ 0.04 pmol͞g with n ϭ 7 for both. Older (20-wk) expressed. These latter proteins are not products of the insulysin adult mice had somewhat higher A␤40 and A␤42 levels than Ϫ Ϫ Ϫ ϩ gene because their presence is not correlated with IDE genotype young (8- to 10-wk) adult mice in all groups (IDE / , IDE / , or with insulysin mRNA or enzyme activity levels. and IDEϩ/ϩ), but similar increases occurred with insulysin To confirm that insulysin activity also was absent from the deficiency. In both age groups, the mean accumulation of both IDEϪ/Ϫ mice, we examined the insulysin-mediated cleavage of the A␤40 and A␤42 peptides (listed in the legend to Fig. 4) was

Miller et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 6223 Downloaded by guest on October 3, 2021 Fig. 4. A␤ peptide accumulation is increased in insulysin-deficient mice. Fig. 3. Brain insulysin mRNA and immunoreactive protein levels are de- A␤40 (A) and A␤42 (B) were quantified by ELISA in brain diethylamine extracts. creased in heterozygote IDEϪ/ϩ mice. (A) Relative insulysin mRNA levels in two Two-way ANOVA revealed a significant effect of both IDE genotype and age sets of mice consisting of one wild-type (WT) and two heterozygote (IDEϪ/ϩ) on the steady-state levels of A␤40 (P Ͻ 0.0001 for both effects) and A␤42 (P Ͻ sex-matched littermates were determined by real-time PCR using the com- 0.001 and P ϭ 0.002, respectively). The post hoc Student–Neuman–Keuls test parative cycle threshold method, assigning a value of 1 to the WT sample in indicated that, for both peptides, the mean values for the three different each set. Insulysin mRNA levels were normalized to 18S rRNA levels in each genotypes were statistically different from each other. In young, adult (8- to sample. (B) Two different quantities of brain homogenate protein (10 and 5 10-wk) IDEϩ/ϩ (n ϭ 3), IDEϩ/Ϫ (n ϭ 3), and IDEϪ/Ϫ (n ϭ 4) mice, the mean A␤40 ␮g) from two IDEϩ/ϩ and two IDEϪ/ϩ mice were resolved by SDS͞PAGE and levels were 1.05, 1.40, and 1.70 pmol͞g, respectively, and the mean A␤42 levels analyzed by Western blotting with the insulysin peptide-specific antibody, were 0.39, 0.48, and 0.56 pmol͞g. In the older (20-wk) IDEϩ/ϩ (n ϭ 2), IDEϩ/Ϫ Ϫ/Ϫ hIDE7–24. The blot is representative of three independent comparisons from (n ϭ 4), and IDE (n ϭ 3) mice, the mean A␤40 levels were 1.36, 1.69, and IDEϪ/ϩ and IDEϩ/ϩ mice. (C) Equal quantities of brain homogenate protein (30 1.97 pmol͞g, respectively, whereas the mean A␤42 levels were 0.46, 0.53, and ␮g) from an IDEϪ/Ϫ mouse and wild-type, IDEϩ/ϩ littermate were resolved by 0.59 pmol͞g. SDS͞PAGE and analyzed by Western blotting with the antibody elicited with recombinant rat insulysin. activity is diminished, but not absent, is of particular note because it suggests that the enzyme activity, rather than substrate significantly different among the three groups (IDEϪ/Ϫ, IDEϪ/ϩ, ␤ ϩ ϩ availability, is rate-limiting in insulysin degradation of A pep- and IDE / ) and inversely correlated with copy number of the tides. Thus, polymorphisms that result in even modest decreases gene-trap allele. in insulysin activity are predicted to result in increased A␤ To examine the influence of insulysin activity on APP and accumulation. Furthermore, the Ϸ50% decrease in insulysin other APP metabolites, we examined the steady-state levels of mRNA, protein, and activity in the heterozygote IDEϩ/Ϫ gene- the APP holoprotein and CTFs. No change in the levels of either trap mice relative to wild-type littermates indicates that there is the APP holoprotein (Fig. 5A)orCTF␤ (not shown) was no compensatory mechanism to increase insulysin expression. detected in the brains of the insulysin-deficient mice relative to The magnitude of A␤ elevation that results from modulation of wild-type littermates. There was, however, a robust, Ϸ6-fold insulysin activity in this model is likely to pose a significant increase in the level of CTF␥, the ␥-secretase-generated increase in the risk of AD. The increase in the A␤ peptide levels C-terminal fragment of APP, in the insulysin-deficient mice in the homozygous IDEϪ/Ϫ mice is similar in magnitude to that (Fig. 5B). seen in presenilin mutation models of AD (19, 20), which increase A␤ production and dramatically accelerate the onset of Discussion AD-like pathology in APP transgenic mice (21, 22). Because of The increase in brain A␤ peptide levels observed in insulysin- the long onset in most cases of sporadic AD, the effect of even deficient mice occurred without any change in the concentration modest increases in A␤ levels accumulated over time also is of APP or CTF␤, the immediate precursor of the A␤ peptides. thought to be highly significant in the development of AD. These findings provide strong evidence that insulysin degrades Insulysin has been shown in vitro to degrade a limited number of A␤ peptides in vivo and contributes to the maintenance of peptides in addition to A␤ (9, 10). Thus, inhibition of insulysin- steady-state levels of A␤ in brain. The increased accumulation of catalyzed A␤ degradation could occur by substrate competition A␤ peptides in heterozygous IDEϪ/ϩ mice in which insulysin during chronic elevation of insulin, ␤-endorphin, or other insu-

6224 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1031520100 Miller et al. Downloaded by guest on October 3, 2021 membrane-bound ectoenzyme and is suggested to be particularly important in the catabolism of extracellular A␤ (25, 26). Insu- lysin is predominately a cytosolic protein, but a portion is targeted to peroxisomes and even smaller amounts are found on the plasma membrane or are secreted (7, 27–29). Thus, in principal, both intracellular and extracellular A␤ could be targets of insulysin-catalyzed A␤ degradation. In CHO cells expressing wild-type human APP, insulysin was found to degrade both nonionic detergent-soluble and detergent-insoluble intracellular A␤ (30). Neprilysin was most active in the degradation of extracellular A␤ and had no activity toward the detergent- soluble intracellular A␤ pool. The additional finding in our studies that CTF␥ levels are increased almost 6-fold in the IDEϪ/Ϫ mice is consistent with the recent report that insulysin can degrade CTF␥ in vitro (31) and extends the in vitro studies to the in vivo situation in the brain. The much greater increase in steady-state levels of CTF␥ in the IDEϪ/Ϫ mice relative to the 1.4- and 1.6-fold increases in the A␤ peptides indicates that insulysin may be more important quan- titatively in CTF␥ degradation. This finding is consistent with the suggestion that insulysin is one of several enzymes responsible for A␤ degradation. Although the function of CTF␥ (also referred to as AICD, APP-intracellular domain) is not yet known, it has been suggested to be a transcriptional regulator (31, 32). Furthermore, smaller, caspase-generated fragments of CTF␥ have been shown to be toxic to neurons in vivo (33–35). Thus, an increase in CTF␥ levels resulting from insulysin deficiency also may represent a risk for neurologic disease. The effects of the accumulation of CTF␥ on brain function and its role in the pathogenesis of AD are important questions for future studies. The insulysin-deficient mouse seems to represent an excellent model system for such studies. Fig. 5. APP levels are unchanged in insulysin-deﬁcient mice, whereas CTF␥ In summary, our studies on the insulysin-deficient mouse fragment levels are elevated. APP (A) and CTF␥ (B) were detected by Western provide strong experimental evidence that two metabolites of blot analysis using the O443 antibody against the C-terminal 20 residues of APP processing, A␤ and CTF␥, are physiologically important APP, and their relative levels were determined by densitometry of the scanned substrates for insulysin and are elevated when insulysin activity images. The values presented for both peptides are the mean Ϯ SEM from ␥ eight IDEϩ/ϩ and six IDEϪ/Ϫ mice. Representative Western blots are shown in C. is diminished. Although the consequences of elevated CTF presently are not understood, based on the large body of evidence linking increased A␤ peptide levels and AD risk, these studies suggest a mechanistic link between changes in lysin substrates, and these conditions also may pose a risk for AD. insulysin activity (genetic or acquired) and an increased risk of Conversely, any increase in insulysin activity may result in the late-onset AD. decreased accumulation of A␤ peptides.

With this report, insulysin joins neprilysin (23) and, most NEUROSCIENCE recently, endothelin-converting enzyme (24) as proteases with a This work was supported in part by grants from the National Institutes of Health (National Institute on Drug Abuse DA 08801 to B.C.M., documented in vivo role in regulating steady-state A␤ peptide ␤ L.B.H., and D.L.T.) and the Alzheimer’s Association (to E.A.E., K.S., levels in brain. It is likely that distinct A pools are catabolized and L.B.H.), a Mayo Alzheimer’s Disease Research Center pilot grant by the different enzymes. Both intracellular and extracellular A␤ (to E.A.E.), and the Mayo Foundation for Medical Education and have been implicated in the etiology of AD. Neprilysin is a Research (to C.B.E.).

1. Hardy, J. & Selkoe, D. J. (2002) Science 297, 353–356. 14. Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C. M., Perez-tur, J., Hutton, M., 2. Sambamurti, K., Greig, N. H. & Lahiri, D. K. (2002) Neuromol. Med. Buee, L., Harigaya, Y., Yager, D., et al. (1996) Nature 383, 710–713. 1, 1–31. 15. Haugabook, S. J., Le, T., Yager, D., Zenk, B., Healy, B. M., Eckman, E. A., 3. Kamal, A., Almenar-Queralt, A., LeBlanc, J. F., Roberts, E. A. & Goldstein, Prada, C., Younkin, L., Murphy, P., Pinnix, I., et al. (2001) FASEB J. 15, 16–18. L. S. (2001) Nature 414, 643–648. 16. Pinnix, I., Musunuru, U., Tun, H., Sridharan, A., Golde, T., Eckman, C., 4. Nunan, J. & Small, D. H. (2000) FEBS Lett. 483, 6–10. Ziani-Cherif, C., Onstead, L. & Sambamurti, K. (2001) J. Biol. Chem. 276, 5. Kurochkin, I. V. & Goto, S. (1994) FEBS Lett. 345, 33–37. 481–487. 6. Qiu, W. Q., Ye, Z., Kholodenko, D., Seubert, P. & Selkoe, D. J. (1997) J. Biol. 17. Affholter, J. A., Fried, V. A. & Roth, R. A. (1988) Science 242, 1415–1418. Chem. 272, 6641–6646. 18. Baumeister, H., Muller, D., Rehbein, M. & Richter, D. (1993) FEBS Lett. 317, 7. Qiu, W. Q., Walsh, D. M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M. B., 250–254. Rosner, M. R., Safavi, A., Hersh, L. B. & Selkoe, D. J. (1998) J. Biol. Chem. 19. Nakano, Y., Kondoh, G., Kudo, T., Imaizumi, K., Kato, M., Miyazaki, J. I., 273, 32730–32738. Tohyama, M., Takeda, J. & Takeda, M. (1999) Eur. J. Neurosci. 11, 2577–2581. 8. Mirsky, I. A. (1957) Recent Prog. Horm. Res. 13, 429–435. 20. Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., 9. Becker, A. B. & Roth, R. A. (1995) Methods Enzymol. 248, 693–703. Wright, K., Saad, I., Mueller, R., Morgan, D., et al. (1998) Nat. Med. 4, 97–100. 10. Safavi, A., Miller, B. C., Cottam, L. & Hersh, L. B. (1996) Biochemistry 35, 21. Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., 14318–14325. Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D., et al. (1996) 11. Zambrowicz, B. P., Friedrich, G. A., Buxton, E. C., Lilleberg, S. L., Person, C. Neuron 17, 1005–1013. & Sands, A. T. (1998) Nature 392, 608–611. 22. Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G., 12. Chomczynski, P. & Saachi, N. (1987) Anal. Biochem. 162, 156–159. Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., et al. (1997) Nat. Med. 3, 13. Nguyen, K. & Miller, B. C. (2002) J. Immunol. 168, 4440–4445. 67–72.

Miller et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 6225 Downloaded by guest on October 3, 2021 23. Iwata, N., Tsubuki, S., Takaki, Y., Shirotani, K., Lu, B., Gerard, N. P., Gerard, 30. Sudoh, S., Frosch, M. & Wolf, B. (2002) Biochemistry 41, 1091–1099. C., Hama, E., Lee, H. J. & Saido, T. C. (2001) Science 292, 1550–1552. 31. Edbauer, D., Willem, M., Lammich, S., Steiner, H. & Haass, C. (2002) J. Biol. 24. Eckman, E. A., Watson, M., Marlow, L., Sambamurti, K. & Eckman, C. B. Chem. 277, 13389–13393. (2003) J. Biol. Chem. 278, 2081–2084. 32. Cao, X. & Sudhof, T. C. (2001) Science 293, 115–120. 25. Iwata, N., Tsubuki, S., Takaki, Y., Watanabe, K., Sekiguchi, M., Hosoki, E., 33. Passer, B., Pellegrini, L., Russo, C., Siegel, R. M., Lenardo, M. J., Schettini, G., Kawashima-Morishima, M., Lee, H. J., Hama, E., Sekine-Aizawa, Y., et al. Bachmann, M., Tabaton, M. & D’Adamio, L. (2000) J. Alzheimers Dis. 2, (2000) Nat. Med. 6, 143–150. 289–301. 26. Fukami, S., Watanabe, K., Iwata, N., Haraoka, J., Lu, B., Gerard, N. P., Gerard, 34. Lu, D. C., Rabizadeh, S., Chandra, S., Shayya, R. F., Ellerby, L. M., Ye, C., Fraser, P., Westaway, D., St. George-Hyslop, P., et al. (2002) Neurosci. Res. X., Salvesen, G. S., Koo, E. H. & Bredesen, D. E. (2000) Nat. Med. 6, 43, 39–56. 397–404. 27. Seta, K. A. & Roth, R. A. (1997) Biochem. Biophys. Res. Commun. 231, 35. Bertrand, E., Brouillet, E., Caille, I., Bouillot, C., Cole, G., Prochiantz, A. & 167–171. 28. Vekrellis, K., Ye, Z., Qiu, W. Q., Walsh, D., Hartley, D., Chesneau, V., Rosner, Allinquant, B. (2001) Mol. Cell. Neurosci. 18, 503–511. M. R. & Selkoe, D. J. (2000) J. Neurosci. 20, 1657–1665. 36. Selkoe, D. J. (2001) Neuron 32, 177–180. 29. Miller, B. C., Thiele, D., Hersh, L. B. & Cottam, G. L. (1996) Immunophar- 37. Carson, J. A. & Turner, A. J. (2002) J. Neurochem. 81, 1–8. macology 31, 151–161. 38. Mukherjee, A. & Hersh, L. B. (2002) J. Alzheimers Dis. 4, 341–348.

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