Miller BC, Eckman EA, Sambamurti K, Dobbs N, Chow KM, Eckman CB, Hersh LB, Thiele DL. Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo. Proc Natl Acad Sci U S A. 2003 May 13;100(10):6221-6. PubMed.

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  1. The newly published paper by Miller et al., well summarized by Tom Fagan above, both confirms and complements our recent study, in which we also utilized IDE gene-disrupted mice to demonstrate that IDE regulates the levels of cerebral Aβ and the AβPP intracellular domain (AICD, CTF in vivo: see Farris et al., 2003). A major strength of this latest work is that by breeding heterozygous IDE knockout mice (IDE+/-) to each other, these authors generated single litters containing all three possible IDE genotypes (IDE+/+, IDE+/-, and IDE-/-). The advantages of this breeding strategy are that it enables one to look for a "gene-dosage" effect on the phenotype of interest while providing littermates of different genotypes for comparison. The disadvantage of this method is that relatively few mice of a given genotype can be generated within one litter. Although the numbers of age-matched animals of a given phenotype in this study were relatively small (n = 2-4), the brain Aβ ELISA data were "tight" enough that statistically significant differences were found.

    First, the authors did a thorough characterization of the three genotypes (standard and real-time RT-PCR, immunoblotting and endorphin degradation assays to measure IDE activity on liver and brain) to demonstrate that the IDE+/- mice do, in fact, have an amount of IDE transcript, protein, and activity that is roughly half that of the IDE+/+ animals. They then convincingly demonstrate a gene-dose response of cerebral Aβ40 and Aβ42. Compared to wild-type mice, those with one copy of the IDE gene had, by my calculations, ~35 percent increase in Aβ40 and ~20 percent increase in Aβ42, while those without any IDE genes had ~55 percent increase in Aβ40 and ~35 percent increase in Aβ42. In addition to strengthening the hypothesis that IDE is responsible for the elevated cerebral Aβ, this gene-dose analysis allows the authors to conclude that IDE activity, rather than substrate availability, is the rate-limiting step in the degradation of Aβ by IDE, and that there is not significant compensatory upregulation of IDE expression by the normal allele when the other is dysfunctional (or, as in this model, absent).

    Of note, although it is inferred that the elevated cerebral Aβ in the IDE+/- and IDE-/- mice is secondary to a deficiency of IDE-mediated Aβ degradation, it was not demonstrated directly in this study that these mice actually have an Aβ degrading deficit. One could hypothesize that the elevated AICD levels in these mice, demonstrated in both studies, could lead to upregulation of AβPP levels. If this increased AβPP was then cleaved by BACE and the γ-secretase complex instead of accumulating in the membrane, Aβ levels could increase without a measurable increase in AβPP—all via a mechanism independent of IDE’s effect on Aβ degradation. To exclude this possibility, we demonstrated a major (>50 percent) Aβ degrading deficit in brain membrane and soluble fractions and intact primary neurons from IDE-/- compared to IDE+/+ mice. We confirmed that the levels of soluble AβPP (AβPPs), released by BACE or γ-secretase cleavage, were indistinguishable between the IDE-/- and IDE+/+ animals, arguing against an increase of Aβ production in the IDE-/- as an explanation for the observed increased levels of cerebral Aβ in these two studies. Additionally, we found that the levels of AβPP and its other proteolytic derivatives (C99, C89, C83), presenilin NTF and CTF, BACE, ADAM 10 and neprilysin were all identical by immunoblotting in the IDE-/- and IDE+/+ mice.

    It is encouraging that the results of the Miller et al. and Farris et al. studies are in quite good agreement regarding IDE’s role in regulating cerebral Aβ and AICD levels in vivo. (It probably should be noted that the cerebral Aβ was extracted by the same DEA protocol and measured using the same ELISA system.) One discrepancy between the papers should be mentioned. A major point of the Farris et al. paper was that selective deletion of one gene, IDE, was not only able to elevate Aβ levels in the brain, but also to recapitulate some of the phenotypic hallmarks of type 2 diabetes, namely hyperinsulinemia and glucose intolerance. We found this particularly exciting in light of the epidemiological evidence suggesting that subjects with type 2 diabetes have approximately a doubling in their risk of Alzheimer’s disease (independent of vascular risk factors), and the emerging genetic evidence for both an Alzheimer’s and type 2 diabetes gene in the IDE region of chromosome 10q (please see paper for more detailed discussion and references). The Miller et al. paper only mentions IDE’s role in insulin/glucose metabolism in one sentence in the Materials and Methods, where they state that they found no difference in blood glucose and insulin levels in fed IDE-/- and IDE+/+ mice, and then suggest that insulin catabolism and activity are not perturbed by the lack of IDE. We demonstrated marked deficiency in insulin degradation by liver fractions, and a significant 2.8-fold elevation in fasting insulin in IDE-/- mice compared to wild-types. The number and age of mice examined and the method of glucose and insulin quantification by Miller et al. is not mentioned, so it is difficult to directly compare experiments, but there may be less of a difference between fed insulin levels in the IDE-/- and IDE+/+ mice, as measured by Miller et al., and fasting insulin levels, as measured by us. We are currently measuring insulin levels in fed animals to see if this may be the case. We found no difference in fasting blood glucose levels between IDE-/- and IDE+/+ mice, but when they were challenged with a glucose load (glucose tolerance test), the IDE-/- animals revealed a significantly delayed drop in blood glucose at 60, 90, and 120 minutes compared to the IDE+/+ mice, showing that the IDE-/- mice are glucose intolerant. Thus, we believe that IDE does have a role in insulin catabolism and glucose tolerance in vivo.

    Taken together, two separate groups have now shown that IDE regulates cerebral levels of Aβ in vivo, making pharmacologic manipulation of protease, either directly or via blocking a natural IDE inhibitor, a potential therapeutic target. The new findings that an abnormally low expression of IDE from one allele is not sufficiently compensated for by the normal allele, and that this partial hypofunction of IDE is sufficient to elevate Aβ levels in the brain, are important. We have recently reported that diabetic GK rats, with their two naturally occurring missense mutations in IDE, show ~30 percent deficit in Aβ degradation in brain fractions and primary neurons, and have markedly elevated levels of endogenously produced Aβ in their neuronal conditioned media (Farris et al., SFN Meeting Abstract, 2002, submitted). Together with the emerging genetic evidence, these findings suggest that IDE mutations could underlie some forms of AD.

    References:

    Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, Eckman CB, Tanzi RE, Selkoe DJ, Guenette S. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):4162-7. PubMed.

    View all comments by Wesley Farris

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