Decreased Catalytic Activity of the Insulin-degrading Enzyme in Chromosome 10-Linked Alzheimer Disease Families
2007; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês
10.1074/jbc.m609168200
ISSN1083-351X
AutoresMinji Kim, Louis B. Hersh, Malcolm A. Leissring, Martin Ingelsson, Toshifumi Matsui, Wesley Farris, Alice Lu, Bradley T. Hyman, Dennis J. Selkoe, Lars Bertram, Rudolph E. Tanzi,
Tópico(s)Biochemical Acid Research Studies
ResumoInsulin-degrading enzyme (IDE) is a zinc metalloprotease that degrades the amyloid β-peptide, the key component of Alzheimer disease (AD)-associated senile plaques. We have previously reported evidence for genetic linkage and association of AD on chromosome 10q23–24 in the region harboring the IDE gene. Here we have presented the first functional assessment of IDE in AD families showing the strongest evidence of the genetic linkage. We have examined the catalytic activity and expression of IDE in lymphoblast samples from 12 affected and unaffected members of three chromosome 10-linked AD pedigrees in the National Institute of Mental Health AD Genetics Initiative family sample. We have shown that the catalytic activity of cytosolic IDE to degrade insulin is reduced in affected versus unaffected subjects of these families. Further, we have shown the decrease in activity is not due to reduced IDE expression, suggesting the possible defects in IDE function in these AD families. In attempts to find potential mutations in the IDE gene in these families, we have found no coding region substitutions or alterations in splicing of the canonical exons and exon 15b of IDE. We have also found that total IDE mRNA levels are not significantly different in sporadic AD versus age-matched control brains. Collectively, our data suggest that the genetic linkage of AD in this set of chromosome 10-linked AD families may be the result of systemic defects in IDE activity in the absence of altered IDE expression, further supporting a role for IDE in AD pathogenesis. Insulin-degrading enzyme (IDE) is a zinc metalloprotease that degrades the amyloid β-peptide, the key component of Alzheimer disease (AD)-associated senile plaques. We have previously reported evidence for genetic linkage and association of AD on chromosome 10q23–24 in the region harboring the IDE gene. Here we have presented the first functional assessment of IDE in AD families showing the strongest evidence of the genetic linkage. We have examined the catalytic activity and expression of IDE in lymphoblast samples from 12 affected and unaffected members of three chromosome 10-linked AD pedigrees in the National Institute of Mental Health AD Genetics Initiative family sample. We have shown that the catalytic activity of cytosolic IDE to degrade insulin is reduced in affected versus unaffected subjects of these families. Further, we have shown the decrease in activity is not due to reduced IDE expression, suggesting the possible defects in IDE function in these AD families. In attempts to find potential mutations in the IDE gene in these families, we have found no coding region substitutions or alterations in splicing of the canonical exons and exon 15b of IDE. We have also found that total IDE mRNA levels are not significantly different in sporadic AD versus age-matched control brains. Collectively, our data suggest that the genetic linkage of AD in this set of chromosome 10-linked AD families may be the result of systemic defects in IDE activity in the absence of altered IDE expression, further supporting a role for IDE in AD pathogenesis. Amyloid β-protein (Aβ) 2The abbreviations used are: Aβ, amyloid β-protein; AD, Alzheimer disease; LOAD, late onset AD; IDE, insulin-degrading enzyme; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSE, neuron-specific enolase. 2The abbreviations used are: Aβ, amyloid β-protein; AD, Alzheimer disease; LOAD, late onset AD; IDE, insulin-degrading enzyme; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSE, neuron-specific enolase. is the primary component of senile plaques, a pathological hallmark in the brains of patients with Alzheimer disease (AD). Elevated levels of cerebral Aβ have also been observed in AD patients (1Selkoe D.J. Neuron. 2001; 32: 177-180Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 2Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Neurology. 2004; 62: 925-931Crossref PubMed Scopus (511) Google Scholar), implicating excessive accumulation of Aβ as a key pathogenic event in AD. Unlike early onset autosomal dominant AD, the vast majority of AD cases do not show any clear evidence of Mendelian transmission and predominantly present with late onset AD (LOAD) (onset age >65). However, there is evidence that genetic factors play a significant role in modifying the disease risk/age of onset in the majority of LOAD cases (3Tanzi R.E. Bertram L. Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar, 4Gatz M. Reynolds C.A. Fratiglioni L. Johansson B. Mortimer J.A. Berg S. Fiske A. Pedersen N.L. Arch. Gen. Psychiatry. 2006; 63: 168-174Crossref PubMed Scopus (1033) Google Scholar). To date, only the ɛ4 allele of the apolipoprotein E gene (APOE) has been firmly established as a LOAD genetic risk factor and has been proposed to be involved in Aβ clearance (5Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3656) Google Scholar). Cerebral Aβ accumulation has been proposed to greatly influence the age of onset of LOAD and is determined by the amount of Aβ generated versus the amount that is degraded and exported from the brain over one's lifetime (6Savage M.J. Trusko S.P. Howland D.S. Pinsker L.R. Mistretta S. Reaume A.G. Greenberg B.D. Siman R. Scott R.W. J. Neurosci. 1998; 18: 1743-1752Crossref PubMed Google Scholar, 7Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (790) Google Scholar).Several proteases have been identified to degrade Aβ, including neprilysin, plasmin, endothelin-converting enzyme-1 as well as insulin-degrading enzyme (IDE) (EC 3.4.24.56) (1Selkoe D.J. Neuron. 2001; 32: 177-180Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). IDE, also called insulysin, is a zinc metalloprotease that cleaves small polypeptides, many of which share amyloid fibril-forming ability, including insulin, atrial naturetic peptide, amylin, calcitonin, and Aβ (8Bennett R.G. Duckworth W.C. Hamel F.G. J. Biol. Chem. 2000; 275: 36621-36625Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9Kurochkin I.V. Trends Biochem. Sci. 2001; 26: 421-425Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). IDE is a major protease to degrade soluble, monomeric Aβ (10Qiu W.Q. Walsh D.M. Ye Z. Vekrellis K. Zhang J. Podlisny M.B. Rosner M.R. Safavi A. Hersh L.B. Selkoe D.J. J. Biol. Chem. 1998; 273: 32730-32738Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 11Vekrellis K. Ye Z. Qiu W.Q. Walsh D. Hartley D. Chesneau V. Rosner M.R. Selkoe D.J. J. Neurosci. 2000; 20: 1657-1665Crossref PubMed Google Scholar) and is localized in the cytoplasm as well as on the cell surface and within mitochondria (11Vekrellis K. Ye Z. Qiu W.Q. Walsh D. Hartley D. Chesneau V. Rosner M.R. Selkoe D.J. J. Neurosci. 2000; 20: 1657-1665Crossref PubMed Google Scholar, 12Leissring M.A. Farris W. Wu X. Christodoulou D.C. Haigis M.C. Guarente L. Selkoe D.J. Biochem. J. 2004; 383: 439-446Crossref PubMed Scopus (132) Google Scholar). Recent studies in animal models have demonstrated that knock out of IDE leads to elevated cerebral Aβ levels along with phenotypic characteristics of type 2 diabetes mellitus (13Farris W. Mansourian S. Chang Y. Lindsley L. Eckman E.A. Frosch M.P. Eckman C.B. Tanzi R.E. Selkoe D.J. Guenette S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4162-4167Crossref PubMed Scopus (1199) Google Scholar, 14Miller B.C. Eckman E.A. Sambamurti K. Dobbs N. Chow K.M. Eckman C.B. Hersh L.B. Thiele D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6221-6226Crossref PubMed Scopus (262) Google Scholar). Conversely, overexpression of IDE attenuates Aβ accumulation in transgenic AD mouse models (15Leissring M.A. Farris W. Chang A.Y. Walsh D.M. Wu X. Sun X. Frosch M.P. Selkoe D.J. Neuron. 2003; 40: 1087-1093Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). Finally, diabetes-inducing mutations in IDE in rat are associated with impaired neuronal Aβ catabolism, supporting co-morbidity of AD, and type 2 diabetes mellitus (16Farris W. Mansourian S. Leissring M.A. Eckman E.A. Bertram L. Eckman C.B. Tanzi R.E. Selkoe D.J. Am. J. Pathol. 2004; 164: 1425-1434Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Several studies have suggested genetic linkage and allelic association between LOAD and the IDE/KIF11 region on chromosome 10q in independent samples (17Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. Selkoe D.J. Saunders A.J. Tanzi R.E. Science. 2000; 290: 2302-2303Crossref PubMed Scopus (455) Google Scholar, 18Edland S.D. Wavrant-De Vriese F. Compton D. Smith G.E. Ivnik R. Boeve B.F. Tangalos E.G. Petersen R.C. Neurosci. Lett. 2003; 345: 21-24Crossref PubMed Scopus (53) Google Scholar, 19Prince J.A. Feuk L. Gu H.F. Johansson B. Gatz M. Blennow K. Brookes A.J. Hum. Mutat. 2003; 22: 363-371Crossref PubMed Scopus (87) Google Scholar, 20Blomqvist M.E. Silburn P.A. Buchanan D.D. Andreasen N. Blennow K. Pedersen N.L. Brookes A.J. Mellick G.D. Prince J.A. Neurogenetics. 2004; 5: 115-119Crossref PubMed Scopus (30) Google Scholar). Our recent report on meta-analyses across all published studies (12 published reports for IDE to date) reveals a significant association of IDE with AD (21Bertram L. McQueen M.B. Mullin K. Blacker D. Tanzi R.E. Nat. Genet. 2007; 39: 17-23Crossref PubMed Scopus (1430) Google Scholar), confirming our original finding on the association and linkage of IDE to AD (17Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. Selkoe D.J. Saunders A.J. Tanzi R.E. Science. 2000; 290: 2302-2303Crossref PubMed Scopus (455) Google Scholar). However, neither pathogenic nor protective IDE gene mutations/variants have yet been validated in AD patients.In an effort to localize novel AD genes, our group previously performed a full-genome linkage screen of the National Institute of Mental Health AD Genetics Initiative family sample (22Blacker D. Bertram L. Saunders A.J. Moscarillo T.J. Albert M.S. Wiener H. Perry R.T. Collins J.S. Harrell L.E. Go R.C. Mahoney A. Beaty T. Fallin M.D. Avramopoulos D. Chase G.A. Folstein M.F. McInnis M.G. Bassett S.S. Doheny K.J. Pugh E.W. Tanzi R.E. Hum. Mol. Genet. 2003; 12: 23-32Crossref PubMed Scopus (303) Google Scholar), which supported our earlier observation of genetic linkage in the chromosomal region near the IDE gene (17Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. Selkoe D.J. Saunders A.J. Tanzi R.E. Science. 2000; 290: 2302-2303Crossref PubMed Scopus (455) Google Scholar). In the current study, we chose three families showing the strongest evidence for linkage to IDE and systematically assessed the activity and expression of IDE in immortalized lymphoblast cell lines from these families. Potential alteration of IDE expression was also investigated in sporadic AD brains to confirm the result from the family-based lymphoblast samples.EXPERIMENTAL PROCEDURESLymphoblastoid Cell Culture—Lymphoblastoid cell lines from affected and unaffected subjects of the three chromosome 10-linked families (total 12 samples) were obtained from The Rutgers University Cell and DNA Repository in New Jersey, and a control lymphoblast (GM7044) unrelated to the AD families was obtained from the Coriell Cell Repository. Among the affecteds, three subjects were male and two were female. The average age at death was 79.7 (±3.9 S.E.) years and the average age of onset was 72.1 (±3.7 S.E.) years. Of the unaffecteds, one subject was male and six were female, with the average age at death of 67.2 (±6.5 S.E.) years. Lymphoblasts were grown in RPMI 1640 medium (BioWhittaker) supplemented with 15% fetal bovine serum (Sigma). After inoculating the same number of living cells, exponentially growing lymphoblasts were collected for analysis.Human Brain Samples—Temporal neocortical tissue from 24 AD brains and 11 control brains without any signs of a neurological disorder were included in the study. All AD subjects had been evaluated at the Memory Disorders Unit at Massachusetts General Hospital and met both the clinical (NINCDS-ADRDA) (23McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan E.M. Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar) and the neuropathological (CERAD, NIA/Reagan) (24Mirra S.S. Heyman A. McKeel D. Sumi S.M. Crain B.J. Brownlee L.M. Vogel F.S. Hughes J.P. van Belle G. Berg L. Neurology. 1991; 41: 479-486Crossref PubMed Google Scholar, 25Hyman B.T. Trojanowski J.Q. J. Neuropathol. Exp. Neurol. 1997; 56: 1095-1097Crossref PubMed Scopus (674) Google Scholar) diagnostic criteria for AD. Nine of the non-neurological control brains had been autopsied at Massachusetts General Hospital, whereas two control brains were from the Harvard Brain Bank and University of Maryland, respectively. Of the AD cases, 58% were male and 42% female. The average age at death was 80.8 years (±1.6 S.E.) with a 15.6-h (±2.8 S.E.) post-mortal interval. Thirteen of the AD cases were APOE-ɛ4-positive (ɛ4+). Among the control subjects, 50% were male and 50% female, with an average age at death of 82.4 years (±1.6 S.E.) years and with a 25.7-h (±5.6 S.E.) post-mortal interval. Two of the control subjects were ɛ4+.In Vitro Insulin Degradation Assay—After collecting lymphoblast cells, membrane and cytosolic fractions were prepared by centrifugation of homogenized cell extracts at 100,000 × g for 1 has described by Huang et al. (26Huang J. Guan H. Booze R.M. Eckman C.B. Hersh L.B. Neurosci. Lett. 2004; 367: 85-87Crossref PubMed Scopus (49) Google Scholar). Insulin-degrading activity was assayed as described by Song et al. (27Song E.S. Juliano M.A. Juliano L. Fried M.G. Wagner S.L. Hersh L.B. J. Biol. Chem. 2004; 279: 54216-54220Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In brief, lymphoblast fractions (0.5–2.0 ml) were incubated with 50 nm tyrosine A14-labeled 125I-insulin (Amersham Biosciences) in 100 mm potassium phosphate buffer (pH 7.3) at 37 °C for 15 min (cytosol) or 120 min (membrane). Intact insulin was then removed by precipitation with 7.5% trichloroacetic acid. Following centrifugation, the degradation products of iodinated insulin, which remain in the supernatant, were counted on a γ counter. Blanks were run with the extract omitted and used for subtracting backgrounds. The degradation rate in pmol/min/mg was calculated from the average of the counts/min values in the trichloroacetic acid supernatant, the specific radioactivity of the iodinated insulin, and the amount of protein in the reaction. The assay was performed in duplicate in all families, whereas four independent measurements were performed for families II and III. The averages were calculated within and across the families.In Vitro Aβ Degradation Assay—Lymphoblast samples were disrupted by incubation in hypotonic buffer (50 mm Tris-HCl, pH 7.4) and extrusion through a 22-gauge hypodermic needle three times. The post-nuclear homogenate was centrifuged at 20,000 × g for 20 min to separate cytosolic (supernatant) and membrane (pellet) fractions, and protein concentration was determined by bicinchoninic acid assay (Pierce). Fluorometric quantification of Aβ degradation was performed using FAβB (fluorescein-Aβ-(1–40)-Lys(LC-biotin)) as described by Leissring et al. (28Leissring M.A. Lu A. Condron M.M. Teplow D.B. Stein R.L. Farris W. Selkoe D.J. J. Biol. Chem. 2003; 278: 37314-37320Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, 5 mg of protein was incubated with 0.5 mm FAβB for 90 min at 20 °C in a degradation buffer (50 mm HEPES, 100 mm NaCl, 10 mm MgCl2, 0.05% bovine serum albumin, pH 7.4). The reactions were quenched by adding avidin to a final concentration of 0.5 mm, and fluorescence polarization (485 excitation, 535 emission) was determined on a PerkinElmer Life Sciences Victor2 multilabel plate reader. Reactions were performed in quadruplicate in the presence and absence of 10 μm insulin, a relatively specific inhibitor of IDE activity, and the percentage of hydrolysis was calculated from standards containing no protease or excess recombinant IDE.RNA Extraction—Total RNA was extracted from lymphoblast samples using the RNeasy kit (Qiagen) as described in the manufacturer's instructions. The concentration of the total RNA was determined using an NP-1000 spectrophotometer (Nanodrop). For brain samples, a 30–50-mg piece of gray matter from the superior temporal sulcus was dissected under stringent RNase-free conditions from each brain in accordance with previously published methods (29Ingelsson M. Ramasamy K. Cantuti-Castelvetri I. Skoglund L. Matsui T. Orne J. Kowa H. Raju S. Vanderburg C.R. Augustinack J.C. de Silva R. Lees A.J. Lannfelt L. Growdon J.H. Frosch M.P. Standaert D.G. Irizarry M.C. Hyman B.T. Acta Neuropathol. (Berl.). 2006; Google Scholar). From each tissue, RNA was extracted with TRIzol reagent according to the manufacturer's instructions (Invitrogen). All samples were controlled for integrity of the 18 and 28 S ribosomal RNAs by microcapillary electrophoresis (not shown) (RNA 6000 Nano Assay, Agilent Technologies), and samples showing degradation were excluded from the study.Reverse Transcription PCR (RT-PCR)—For the lymphoblast samples, first-strand cDNAs were synthesized from 5 μg of the total RNA using 200 units of Superscript III reverse transcriptase (Invitrogen) and random hexamers as described in the manufacturer's instructions. To investigate alternatively spliced transcript variants, RT-PCR was carried out using the cDNAs as templates with TaqDNA polymerase (Qiagen) or Turbo Pfu DNA polymerase (Stratagene) and corresponding primers (Table 1). For the brain tissue samples, reverse transcription was carried out on 2 μg of all total RNA samples to generate an equal number of cDNA copies using random hexamers and 200 units of Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions.TABLE 1Primers and a probe used in this studyNameSequenceIDE TaqMan probe5′-ATGCAGCCTGCTTTGTT-3′IDE forward primer-TaqMan5′-GAAGCCCTTCTCCATGGAAACAT-3′IDE reverse primer-TaqMan5′-AGGGTGTCTTCAACCATCTGCATAA-3′IDE 1F5′-GCAAGCAGGAAGCGTTTG-3′IDE 5R5′-CAATGCCTTCTTGGTTTGGT-3′IDE 6F5′-CCTGAACACCCTTTCCAAGA-3′IDE 13R5′-AAGAGCAGGGTATGGTGTCG-3′IDE 14F5′-CCGAAGGCTTGTCTCAACTT-3′IDE 15a-R5′-ATACATCCCATAGATGGTATTTTGG-3′IDE 15b-R5′-TGCATTCATTCCTGATGCAATGC-3′IDE 17R5′-GCCACTTCAGTCATCAGCAA-3′IDE 19R5′-AACCAGCTGACTTGGAAGGA-3′IDE 20F5′-GAGGATGGTTTGTTTATCAGCAG-3′IDE 25R5′-AAAGTGGCCAAGATGATTTTC-3′GAPDH forward primer5′-GGTCTCCTCTGACTTCAACA-3′GAPDH reverse primer5′-GTGAGGGTCTCTCTCTTCCT-3′NSE forward primer5′-CGCCACTACCACCGTCTGA-3′NSE reverse primer5′-TAGAGGCTCCACTGGGCACTG-3′ Open table in a new tab Quantitative RT-PCR—All quantitative PCR analyses were performed with iCycler™ (Bio-Rad). To determine IDE mRNA levels in the lymphoblast and brain samples, a 6-carboxyfluorescein-labeled TaqMan probe and a set of PCR primers (forward and reverse primers-TaqMan, Table. 1) were synthesized (Applied Biosystems), and quantitative PCR was performed on equal amounts of cDNA from each sample using TaqMan universal PCR master mix (Applied Biosystems). The IDE mRNA levels were measured in triplicates with four independent experiments and determined by the standard curve method. Relative IDE mRNA levels were calculated by normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or neuron-specific enolase (NSE, ENO2) mRNA levels, which were generated by quantitative PCR on the same cDNA samples using the SyBR Green I detection system (Applied Biosystems). Forward and reverse primers for GAPDH and NSE were designed with Primer Express™ software (PE Applied Biosystems) and synthesized (Qiagen). Each PCR product was sequenced to confirm its identity. For levels of exons 15a- and 15b-specific IDE mRNAs in the lymphoblast samples, a common forward primer residing in exon 14 (IDE 14F in Table 1) and either of the reverse primers specific for exon 15a or 15b were used for SyBR Green quantitative RT-PCR. The quantitative RT-PCR was done in triplicates with three independent experiments. Each exon-specific amplified DNA fragment was confirmed by sequencing. The pCR2.1 vectors (Invitrogen) harboring the same PCR fragments with exons 15a or 15b were utilized to generate standard curves. All of the PCR primer pairs for quantitative RT-PCR were located in different exons to avoid possible amplification of genomic DNA. Sequences of all of the primers and the probe are listed in Table 1.Western Blot Analysis—Total cell extracts were obtained by resuspending lymphoblast cells in radioimmune precipitation assay buffer (10 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% cholic acid, 0.1% SDS, and 5 mm EDTA). The total lysates were separated on 4–12% BisTris gel (Invitrogen), transferred to immunoblot polyvinylidene difluoride membrane (Bio-Rad), and hybridized with IDE polyclonal antibody (IDE-1, (11Vekrellis K. Ye Z. Qiu W.Q. Walsh D. Hartley D. Chesneau V. Rosner M.R. Selkoe D.J. J. Neurosci. 2000; 20: 1657-1665Crossref PubMed Google Scholar)) and actin monoclonal antibody (pan Ab-5, Neo-Markers). Quantification of the chemiluminescence signal was accomplished using the VersaDoc imaging system and the Quantity One quantification program (Bio-Rad). Relative IDE protein levels were calculated by normalization of IDE levels with actin signals, and averages from four independent experiments were taken.RESULTSIDE Activity in the Chromosome 10-Linked AD Families—We first assessed IDE activity in the lymphoblast cell lines derived from 12 subjects of three AD families exhibiting particularly strong evidence of genetic linkage to chromosome 10 in the IDE gene region (17Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. Selkoe D.J. Saunders A.J. Tanzi R.E. Science. 2000; 290: 2302-2303Crossref PubMed Scopus (455) Google Scholar, 22Blacker D. Bertram L. Saunders A.J. Moscarillo T.J. Albert M.S. Wiener H. Perry R.T. Collins J.S. Harrell L.E. Go R.C. Mahoney A. Beaty T. Fallin M.D. Avramopoulos D. Chase G.A. Folstein M.F. McInnis M.G. Bassett S.S. Doheny K.J. Pugh E.W. Tanzi R.E. Hum. Mol. Genet. 2003; 12: 23-32Crossref PubMed Scopus (303) Google Scholar). Because insulin possesses a high affinity for IDE and is a major substrate of this peptidase, we employed a sensitive and reliable insulin degradation assay to assess IDE catalytic activity in membrane and cytosolic fractions prepared from lymphoblast samples (Fig. 1A). Peptidolytic activity of cytosolic IDE was significantly decreased in the affected versus unaffected subjects (>50%; p = 0.001) after combining the members of all three families. Additionally, in one family (I), cytosolic activity of IDE was significantly lower in three affected versus three unaffected subjects (p = 0.008). IDE activity was also decreased in the membrane fractions of the affected (versus unaffected) individuals; however, these differences did not reach statistical significance.To confirm our finding of decreased cytosolic IDE activity particularly in families I and III, affecteds of which exhibited the most robust decreases in IDE activity in the insulin degradation assay, peptidolytic activity of IDE was next assayed using Aβ1–40 as a substrate (Fig. 1B). IDE-mediated degradation of Aβ was decreased in affected versus unaffected subjects in both families. However, this difference did not reach statistical significance at least partly because of the less sensitive nature of this assay (versus the insulin-degrading assay) and lower affinity of IDE for Aβ. Meanwhile, we could not obtain reliable data for the Aβ-degrading ability of membrane IDE, possibly because of the limiting amount of IDE in the membrane fractions and decreased affinity of IDE for Aβ.IDE Expression in the Chromosome 10-Linked AD Families—We next assessed IDE expression by performing quantitative RT-PCR on cDNAs generated from lymphoblast cell lines from the same three AD families. The mRNA levels of IDE, normalized to GAPDH, were highly variable across samples and revealed no significant or consistent differences among affected versus unaffected subjects across the families (Fig. 2, A and B, top). The families also exhibited differing trends for IDE mRNA levels in affected versus unaffected individuals; e.g. in family I, affected subjects had lower IDE mRNA levels than the unaffecteds (p = 0.022), whereas an opposite trend was observed in family III (p = 0.020).FIGURE 2Expression of IDE in the chromosome 10-linked AD families. IDE mRNA levels were measured in lymphoblast samples from each member of the chromosome 10-linked AD families by quantitative RT-PCR and normalized with GAPDH mRNA levels (top). IDE protein levels were also determined by quantitative Western blot analysis and normalized with actin protein signals (bottom). A, graphs of the individual samples. Results are shown as mean ± S.D. of four independent experiments. Roman numbers indicate different AD families. C, control lymphoblast (GM7044); U, unaffected; A, affected. B, tables of averages within and across families. Asterisks denote results with p < 0.05. p values were calculated by two-tailed Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, IDE protein levels were determined by quantitative Western blot analysis of total protein extract (Fig. 2, A and B, bottom). Relatively high IDE expression in affected versus unaffected subjects of family III was confirmed at the protein level (p = 0.018). However, the affected subjects in family I, who had relatively low IDE message levels, revealed no significant reductions in IDE protein levels. Overall, protein levels of IDE across the families did not exhibit significant or consistent differences according to disease status.Levels of Alternative IDE Transcripts in the Chromosome 10-Linked AD Families—After establishing that no IDE coding mutations existed in the canonical exons of the IDE gene in these AD families, we next explored the possibility that altered mRNA splicing of IDE may account for the observed reduction in IDE activity. First, Northern blot analysis was performed with total RNA extracted from the lymphoblast samples. Two different IDE transcripts were detected (data not shown) that had similar sizes to those previously reported in rat, 3.6 and 5.9 kb (30Baumeister H. Muller D. Rehbein M. Richter D. FEBS Lett. 1993; 317: 250-254Crossref PubMed Scopus (67) Google Scholar). Northern blotting using PCR fragments covering three different IDE coding regions (Fig. 3A, a), (b), and (d)) as probes, detected the two IDE mRNAs of indistinguishable sizes with no other additional bands (data not shown). Recently, Farris et al. (31Farris W. Leissring M.A. Hemming M.L. Chang A.Y. Selkoe D.J. Biochemistry. 2005; 44: 6513-6525Crossref PubMed Scopus (73) Google Scholar) detected up to four different Northern blot bands of IDE in different tissues, two of which corresponded in size with the transcripts found in our lymphoblast samples. Based on the previously reported observations, we suspect that the two major IDE messages observed in these lymphoblasts are due to alternative polyadenylation sites. In any event, the two Northern blot bands were coordinately expressed, suggesting no significant differential regulation of the two transcripts among lymphoblasts from these families.FIGURE 3Levels of alternative IDE transcripts in the chromosome 10-linked AD families. A, diagram of 25 conventional IDE exons with conserved domains of peptidase M16 family. PCR primers (arrows) and each RT-PCR product (dashed lines) are indicated. B, RT-PCR was performed on total RNA extracted from a lymphoblast sample of each AD family member with the four different primer pairs spanning the coding region of IDE. RT-PCR products were separated on 2% agarose gel. The expected sizes of RT-PCR products are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess alternative splicing of IDE, we performed RT-PCR on total RNA obtained from each lymphoblast sample using oligonucleotide primer pairs spanning the entire IDE coding region (Fig. 3A). As shown in Fig. 3B, no changes in sizes of PCR products were observed, suggesting no evidence for alternative splicing in the major transcript in these samples. Subsequently, full-length IDE cDNA was produced by RT-PCR using high fidelity DNA polymerase (Pfu, Stratagene), and possible sequence changes in IDE mRNA were investigated more closely by sequencing. Neither exon swapping between similar-sized exons nor nucleotide sequence changes was observed in the major
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