Insulin-degrading enzyme: structure-function relationship and its possible roles in health and disease.

Insulin-degrading enzyme (IDE) or insulysin is a highly conserved Zn(2+) -dependent endopeptidase with an "inverted" HxxEH motif. In vivo, IDE contributes to regulate the steady state levels of peripheral insulin and cerebral amyloid beta peptide (Abeta) of Alzheimer's disease. In vitro, substrates of IDE include a broad spectrum of peptides with relevant physiological functions such as atrial natriuretic factor, insulin-like growth factor-II, transforming growth factor-alpha, beta-endorphin, amylin or glucagon. The recently solved crystal structures of an inactive IDE mutant bound to four different substrates indicate, in accordance with previous compelling biochemical data, that peptide backbone conformation and size are major determinants of IDE recognition and substrate selectivity. IDE-N and IDE-C halves contribute to substrate binding and may rotate away from each other leading to open and closed conformers that permit or preclude the entry of substrates. Noteworthy, stabilization of substrate beta strands in their IDE-bound form may explain the preference of IDE for peptides with a high tendency to self-assembly as amyloid fibrils. These structural requirements may underlie the capability of some amyloid peptides of forming extremely stable complexes with IDE and raise the possibility of a dead-end chaperone-like function of IDE independent of catalysis. Furthermore, the recent recognition of IDE as a varicella zoster virus receptor and its putative involvement in muscle cell differentiation, steroid receptor signaling or proteasome modulation suggest that IDE is a multi-functional protein with broad and relevant roles in several basic cellular processes. Accordingly, IDE functions, regulation or trafficking may partake in the molecular pathogenesis of major human diseases and become potential targets for therapeutic intervention.

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis.

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.

Defective ubiquitination of cerebral proteins in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid-beta. Ubiquitin has also been shown to be present in some of the inclusions characteristic of this disease. To obtain further insight into the role played by the ubiquitin pathway in AD, we investigated the capacity of postmortem samples of cerebral cortex from normal and AD patients to form high-molecular-weight ubiquitin-protein conjugates. Activity of the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2) involved in the ubiquitin pathway was also determined. In normal samples, the amount of high-molecular-weight ubiquitin-protein conjugates (HMW-UbPC) in cytosol increased with incubation time, whereas, in samples of AD cases, these were almost undetectable. The addition of an adult rat fraction, enriched in ubiquitinating enzymes, restored the capacity of AD brain cytosolic fraction to form conjugates. The trypsin-like proteolytic activity of the 26S proteasome was found to be decreased in AD cytosol brain. Assay of the activity of E1 and E2 by thiol-ester formation revealed a significant decrease in AD samples. Moreover, Western blotting using a specific antibody against E1 showed a dramatic drop of this enzyme in the cytosolic fraction, whereas normal levels were found in the particulate fraction, suggesting a possible delocalization of the enzyme. Our results suggest that a failure in the ubiquitination enzymatic system in brain cytosol may contribute to fibrillar pathology in AD.

Degradation of soluble amyloid beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains.

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid beta peptide (A(beta)) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A(beta) analogs 1-40, 1-42 and the Dutch variant 1-40Q at physiological concentrations (1 nM). The rate of synthetic 125I-A(beta) degradation was similar among the A(beta) analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either 125I-insulin, 125I-Abeta 1-40 or 125I-A(beta) 1-42 and both A(beta) degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A(beta) analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 +/- 0.4, 2.0 +/- 0.1 and 2.3 +/- 0.3 microM for A(beta) 1-40, A(beta) 1-42 and A(beta) 1-40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A(beta) degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A(beta) accumulation in the brain.

Differential accumulation of soluble amyloid beta peptides 1-40 and 1-42 in human monocytic and neuroblastoma cell lines. Implications for cerebral amyloidogenesis.

Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid beta protein (A(beta)). While A(beta)1-40 predominates in the vascular system, A(beta)1-42 is the major component of the senile plaques in the neuropil. The concentration of both A(beta) species required to form amyloid fibrils in vitro is micromolar, yet soluble A(betas) found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of A(beta) sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of A(beta), we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125I-A(beta)1-40 and 125I-A(beta)1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of A(beta) in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125I-A(betas) than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125I-A(beta)1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125I-A(beta)1-40. These results support that A(beta)1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar A(beta)1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.

Internalization and resistance to degradation of Alzheimer's A beta 1-42 at nanomolar concentrations in THP-1 human monocytic cell line.

Microglial cell involvement in Alzheimer's disease has been related to amyloid beta (A beta) internalization, the release of inflammatory cytokines and the development of neuritic plaques. The human monocyte/macrophage THP-1 cell line has been widely used as a model of human microglial cells. We used THP-1 cells to study the adsorption, internalization and resistance to degradation of A beta1-40 and A beta1-42 isoforms offered at nanomolar concentrations and free of large aggregates, conditions that may mimic a pre-fibrillar stage of A beta in the brain. Under these conditions, A betas did not induce THP-1 activation, as assessed by interleukin-1beta expression. A beta1-42 showed a preferential adsorption and intracellular accumulation as compared to A beta1-40, supporting that competent nuclei for A beta1-42 ordered aggregation may be formed inside microglial cells. In light of the possible neurotoxicity of soluble A beta1-42, we propose that amyloid formation within brain phagocytic cells may be a protective mechanism in early stages of the disease.

Presenilin 1 Met146Leu variant due to an A --> T transversion in an early-onset familial Alzheimer's disease pedigree from Argentina.

Most of the cases of early-onset familial Alzheimer's disease (FAD) are related to missense mutations in the presenilin 1 (PS-1) gene on chromosome 14. Although PS-1 mutations are distributed throughout the entire open reading frame, most mutations are found in transmembrane region II and hydrophilic loop VI encoded by exons 5 and 8, respectively. These two groups of substitutions are associated with an age of onset of 40-43 years for exon 5 and 45-55 years for exon 8, respectively. We have previously described a South American pedigree from Argentina with early-onset FAD (mean age of onset 38.9 +/- 3.9 years) with no mutations in exons 16 and 17 of the beta-protein precursor gene (betaPP770 transcript). Here we report the identification of an A --> T transversion at the first position of codon 146 of PS-1 in these patients. This missense mutation results in a Met --> Leu substitution, as reported for the Italian pedigrees Tor1.1 and FAD4. The significant differences in ages of onset and death among members of generations II-III and IV suggest that other genetic and/or environmental factors may influence disease phenotype in this pedigree.

Early onset Alzheimer's disease in a South American pedigree from Argentina.

We report the clinical, SPET, immunohistochemical and DNA features of an early-onset familial Alzheimer's disease (FAD) in an Argentine pedigree of South American indian ethnic background. Pedigree spans 5 generations comprising more than 110 biological relatives. Clinical data supported the diagnosis of early onset FAD (mean age at onset 38.9 years) in 10 family members, including 3 with pathological confirmation (mean age at death 48.5). The pattern of transmission suggested autosomal dominant inheritance. Prominent features were mood changes, early language impairment, myoclonus, seizures and cerebellar signs. SPET displayed bilateral frontal, temporo-parietal and cerebellar hypoperfusion in early stages and in an asymptomatic member at risk, suggesting that SPET may have predictive value in this family. Immunohistochemistry showed beta amyloid deposits within neuritic plaques and vessel walls and no anti-PrP immunoreactivity. DNA analysis showed no abnormalities in the beta amyloid precursor protein gene. The identification of additional genetic defects in well characterized independent FAD pedigrees will contribute to the understanding of the pathogenesis of Alzheimer's disease.