Suzanne Y. Guénette

Insulin-degrading enzyme regulates the levels of insulin, amyloid β-protein, and the β-amyloid precursor protein intracellular domain in vivo

Two substrates of insulin-degrading enzyme (IDE), amyloid β-protein (Aβ) and insulin, are critically important in the pathogenesis of Alzheimers disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Aβ levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Aβ and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE −/−). IDE deficiency resulted in a >50% decrease in Aβ degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE −/− mice showed increased cerebral accumulation of endogenous Aβ, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the β-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.

Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development

Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP‐dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins.

hFE65L Influences Amyloid Precursor Protein Maturation and Secretion

Abstract : The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective α‐secretase‐derived secreted APP (APPsα) and the Alzheimer’s disease‐associated β‐amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C‐terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin‐coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a threefold increase in the amount of APPsα recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPsα secreted is due neither to a simple increase in the steady‐state levels of APP nor to activation of the protein kinase C‐regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.

Generation of the β-Amyloid Peptide and the Amyloid Precursor Protein C-terminal Fragment γ Are Potentiated by FE65L1

Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of α-secretase-derived APP (APPsα). Increased β-amyloid (Aβ) generation was also observed in cells showing the FE65-dependent increase in APPsα. To understand the mechanism for the observed increase in both Aβ and APPsα given that α-secretase cleavage of a single APP molecule precludes Aβ generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates γ-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and Aβ40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on γ-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

FE65 Interaction with the ApoE Receptor ApoEr2

The adaptor protein FE65 interacts with the β-amyloid precursor protein (APP) via its C-terminal phosphotyrosine binding (PTB) domain and affects APP processing and Aβ production. Our previous data demonstrate that the apoE receptor ApoEr2 co-precipitated with APP and suggest that there are extracellular and intracellular interactions between these two transmembrane proteins. We hypothesized that FE65 acts as an intracellular link between ApoEr2 and APP. Co-immunoprecipitation experiments in COS7 cells demonstrated an interaction between ApoEr2 and FE65 that depended on the N-terminal PTB domain of FE65. Full-length FE65 increased co-immunoprecipitation of ApoEr2 and APP. Full-length FE65 also increased surface expression of ApoEr2, as determined by surface protein biotinylation and live cell surface staining. Constructs containing both the C- and N-terminal PTB domains of FE65 increased secreted APP, secreted ApoEr2, APP C-terminal fragment, and ApoEr2 C-terminal fragment, but constructs containing only single PTB domains did not affect APP or ApoEr2 processing. In addition, full-length FE65 decreased Aβ to a significantly greater extent than individual FE65 domains. These data suggest that FE65 can bind APP and ApoEr2 at the same time and affect the processing of each.

HtrA2 Regulates β-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

Alzheimer disease-associated β-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Aβ secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

Candidate genes showing no evidence for association or linkage with Alzheimer's disease using family-based methodologies

Alzheimers disease (AD) is a genetically complex and heterogeneous disorder. To date, a large number of candidate genes have been associated with the disease, however none of these findings has been consistently replicated in independent datasets. In this study we report the results of family-based analyses for polymorphisms of five such candidates on chromosomes 2 (interleukin-1beta, IL-1B), 3 (butyrylcholinesterase, BCHE), 11 (cathepsin D, CTSD; Fe65, APBB1) and 12 (lipoprotein receptor-related protein-1, LRP1) that were all suggested to be associated with AD in recent case-control studies. To minimize the possibility of spurious findings due to population admixture, we used a family-based design applying the sibship disequilibrium test (SDT) as well as two-point parametric linkage analyses on families from the National Institute of Mental Health (NIMH) Genetics Initiative. Contrary to the initial reports, none of the polymorphisms that were analyzed showed evidence for association or linkage with AD in our families. Our results suggest that the previously reported associations from case-control studies are either (a) false positive results, e.g. due to type I error or population admixture, (b) smaller than initially proposed, or (c) due to linkage disequilibrium with an as yet unidentified polymorphism nearby.

Astrocytes: a cellular player in Aβ clearance and degradation

Abstract Astrocytes are thought to play a protective role in Alzheimers disease (AD) by shielding neurons from the toxic effects of extracellular senile plaques. The mechanisms involved in the recruitment of astrocytes to senile plaques are unclear, and studies examining the astrocyte response to extracellular β-amyloid (Aβ) are limited. However, recent work has shown that astrocytes migrating towards monocyte chemoattractant protein-1 become immobilized when they encounter Aβ. Once in the presence of Aβ, astrocytes can internalize and degrade it. It is possible that this proposed function for astrocytes is inactivated or overwhelmed in AD.

No evidence for genetic association or linkage of the cathepsin D (CTSD) exon 2 polymorphism and Alzheimer disease

Two recent case‐control studies have suggested a strong association of a missense polymorphism in exon 2 of the cathepsin D gene (CTSD) and Alzheimer disease (AD). However, these findings were not confirmed in another independent study. We analyzed this polymorphism in two large and independent AD study populations and did not detect an association between CTSD and AD. The first sample was family‐based and included 436 subjects from 134 sibships discordant for AD that were analyzed using the sibship disequilibrium test (SDT, p = 0.68) and the sib transmission/disequilibrium test (Sib‐TDT, p = 0.81). The second sample of 200 AD cases and 182 cognitively normal controls also failed to show significant differences in the allele or genotype distribution in cases versus controls (X2, p = 0.91 and p = 0.88, respectively). In addition, two‐point linkage analyses in an enlarged family sample (n = 670) did not show evidence for linkage of the chromosomal region around CTSD. Thus, our analyses on more than 800 subjects suggest that if an association between the CTSD exon 2 polymorphism and AD exists, it is likely to be smaller than previously reported. Ann Neurol 2001;49:114–116

Mechanisms of Aβ clearance and catabolism

Mutations that result in an increased generation of amyloid beta peptide (Aβ) account for less than 5% of Alzheimer’s disease (AD). Data suggesting that late onset AD risk factors play a role in Aβ turnover in the brain have shifted some of the research focus to the study of Aβ clearance and degradation and the impact of these processes on the etiology of Alzheimer’s disease (AD). This review will examine the data obtained from studies performed in knockout and transgenic mice on the proteases; the cells and the physiological mechanisms that play a part in the removal of Aβ from the brain.