Abstract
Insulin-degrading enzyme (IDE) is a zinc metalloprotease that degrades the amyloid beta-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.
Highlights
insulin-degrading enzyme (IDE) in Chromosome 10-Linked Alzheimer disease (AD) Families tute of Mental Health AD Genetics Initiative family sample [22], which supported our earlier observation of genetic linkage in the chromosomal region near the IDE gene [17]
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)
IDE-mediated degradation of A was decreased in affected versus unaffected subjects in both families
Summary
Lymphoblastoid 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. 50% were male and 50% female, with an average age at death of 82.4 years (Ϯ1.6 S.E.) years and with a 25.7-h (Ϯ5.6 S.E.) post-mortal interval. 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. Reverse Transcription PCR (RT-PCR)—For the lymphoblast samples, first-strand cDNAs were synthesized from 5 g of the total RNA using 200 units of Superscript III reverse transcriptase (Invitrogen) and random hexamers as described in the manufacturer’s instructions. For the brain tissue samples, reverse transcription was carried out on 2 g 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. 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 syn-
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