Using genetics to enable studies on the prevention of Alzheimer's disease.

Curing Alzheimer's disease (AD) remains an elusive goal; indeed, it may even prove to be impossible, given the nature of the disease. Although modulating disease progression is an attractive target and will alleviate the burden of the most severe stages, this strategy will not reduce the prevalence of the disease itself. Preventing or (as described in this article) delaying the onset of cognitive impairment and AD will provide the greatest benefit to individuals and society by pushing the onset of disease into the later years of life. Because of the high variability in the age of onset of the disease, AD prevention studies that do not stratify participants by age-dependent disease risk will be operationally challenging, being large in size and of long duration. We present a composite genetic biomarker to stratify disease risk so as to facilitate clinical studies in high-risk populations. In addition, we discuss the rationale for the use of pioglitazone to delay the onset of AD in individuals at high risk.

A TOMM40 variable-length polymorphism predicts the age of late-onset Alzheimer's disease.

The ɛ4 allele of the apolipoprotein E (APOE) gene is currently the strongest and most highly replicated genetic factor for risk and age of onset of late-onset Alzheimer's disease (LOAD). Using phylogenetic analysis, we have identified a polymorphic poly-T variant, rs10524523, in the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene that provides greatly increased precision in the estimation of age of LOAD onset for APOE ɛ3 carriers. In two independent clinical cohorts, longer lengths of rs10524523 are associated with a higher risk for LOAD. For APOE ɛ3/4 patients who developed LOAD after 60 years of age, individuals with long poly-T repeats linked to APOE ɛ3 develop LOAD on an average of 7 years earlier than individuals with shorter poly-T repeats linked to APOE ɛ3 (70.5 ± 1.2 years versus 77.6 ± 2.1 years, P=0.02, n=34). Independent mutation events at rs10524523 that occurred during Caucasian evolution have given rise to multiple categories of poly-T length variants at this locus. On replication, these results will have clinical utility for predictive risk estimates for LOAD and for enabling clinical disease prevention studies. In addition, these results show the effective use of a phylogenetic approach for analysis of haplotypes of polymorphisms, including structural polymorphisms, which contribute to complex diseases.

The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.

The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 was able to bind Pin1, an event we have recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1.

Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. I. In vitro studies.

The function of the hydrophilic carboxyl-terminal region of Drosophila DNA topoisomerase II was examined by constructing a series of deletion mutants at the 3'-end of the Drosophila Top2 cDNA. The truncated enzymes were then expressed in Saccharomyces cerevisiae. Deletion of up to 240 out of 1447 total amino acids had no apparent effect on the enzyme's ability to catalyze topisomerization reactions. When 273, or more, amino acids were deleted, the enzyme was no longer active. Examples were found where deletion of less than 240 amino acids inactivated the enzyme. Based on the hydrodynamic properties determined for one of these mutants, the lack of activity was most likely due to misfolding of the polypeptides. The active mutants have similar hydrodynamic properties and heat inactivation profiles as the intact enzyme, suggesting that they are dimeric and stably folded. The carboxyl-terminal 240 amino acids also were not required for interaction with the drug VM26. The only difference noted between the shortest, active mutant and the full-length enzyme was a decrease in the stability of the interaction of the truncated enzyme with DNA as evidenced by a decrease in the ionic strength at which catalysis was optimal and at which the transition between a processive and distributive mode of supercoil relaxation occurred.

Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. II. In vivo studies.

Genetic complementation, protein distribution, and in vivo enzymatic activity by carboxyl-terminal truncation mutations of the Drosophila enzyme were examined. Removal of more than 273 of the 1447 amino acids composing the full-length topoisomerase inactivates the enzyme in vivo and in vitro; removal of 227 amino acids or less has no apparent effect on the ability of the enzyme to substitute for a conditional lethal, or null mutation, of the Saccharomyces cerevisiae top2 gene. Four catalytically active mutants, from which 227 or 240 amino acids are deleted, define an intervening, critical region. Each mutant in this critical region displays different in vivo complementation activity ranging from complete complementation to noncomplementation. Deletion analysis revealed a potent nuclear localization signal within the most distal 60 amino acids, although this is apparently not the only functional signal sequence encoded in the enzyme. Subcellular fractionation and indirect immunofluorescence demonstrate that the truncated enzymes localize to the nucleus, albeit with reduced efficiency compared to wild type. The ability of these mutants, including a mutant in the critical region which does not complement, to catalyze the decatenation of replicated plasmids and the segregation of replicated chromosomes was also examined.