The Basic Biology of Aging at the University of Washington
Toby Cole

Email:tobycole@u.washington.edu

Research by Dr. Cole on the paraoxonase family of proteins (PON1, PON2, and PON3) has been productive in the past year, resulting in two published manuscripts and presentation of the findings at several national meetings. He succeeded in the last year in expressing active recombinant PON1 in E. coli, and he is using three different approaches to obtain purified recombinant PON1 without a fusion domain attached. PON2 was also successfully expressed in E. coli, and we are developing an assay to determine whether it is active. This will accelerate progress in obtaining sufficiently large crystals for x-ray diffraction, and also allows us to generate variants with altered activity for specific substrates, with the aim of understanding the molecular structural basis of the PON1Q192R activity polymorphism. We have also been investigating the factors that affect PON1 expression, in an attempt to understand the widely different levels of expression between individuals. Recently, we encouraged Dr. Debbie Nickerson (Seattle SNPs Program) to explore the genetic diversity of the PON1 and PON2 genes by sequencing the entire PON1 and PON2 genes of 47 individuals. The results provided a wealth of new information about additional polymorphisms that explain why some individuals have altered PON1 activity, and also provided the gene frequency for the PON1192 polymorphism in the black subpopulation. Interestingly, there is essentially a complete reversal of the position 192 allele frequencies between Northern European populations (Q = 0.7; R = 0.3) and the CEPH black subpopulation sample (Q = 0.3; R = 0.7). In a separate study, because the position –108 polymorphism was observed to have the most dramatic effect on PON1 expression levels in our population studies and this site represents a consensus SP1 binding site, we used a chip-based surface plasmon resonance (SPR) sensor system developed in our laboratory to examine binding of recombinant SP1 to the position –108 binding site. We observed specific binding of recombinant SP1 to this site, supporting the conclusion that the -108CT polymorphism occurs at an important SP1 binding site.

Progress on development of the PON1Q192R transgenic mouse model has provided important insights into the in vivo function of the human PON1192 alloforms. We now have robust colonies of both hPONR192 and hPONQ192 mice on the PON1-/- background. The transgenic strains are expressing human plasma PON1 with the expected properties of each of the respective human PON1192 alloforms. We recently completed a set of experiments to assess the conservation of factors that regulate the developmental expression of PON1. We followed the expression of the human PON1 genes (under the regulation of the human 5' PON1 regulatory sequences) in the two transgenic mouse lines. We found that the expression of human PON1 in these mice followed the mouse timeline of expression, peaking at about 21 days. These results indicate that there is a high degree of conservation between the factors that regulate PON1 expression between humans and mice. We also followed the developmental time course of appearance of PON1 in the plasma of 9 human individuals (5 Q/Q and 4 R/R). Differences were observed among individuals with respect to the time at which their plasma PON1 levels approached a plateau level (6 to ~15 mos). This work has now been published in Pharmacogenetics. We are currently measuring PON1 activity in the plasma of these same transgenic mice as they age.

In addition to the work on paraoxonases, the Genetic Approaches to Aging Training Grant helped to support continuing research on the involvement of zinc in amyloid plaque formation in a mouse model of Alzheimer’s Disease. The preliminary work on zinc described briefly in the original training grant proposal resulted in a 2002 publication in Proc Natl Acad Sci (under other support) demonstrating the importance of zinc in amyloid plaque deposition. This work has now been extended to demonstrate that zinc released at the synapse contributes to beta-amyloid deposition in the cerebral vasculature as well, leading to Cerebral Amyloid Angiopathy (CAA). This work has been submitted to the Journal of Neuroscience, with cited support from the Genetic Approaches to Aging training grant.

Publications:
1. Cole TB, Jampsa RL, Walter BJ, Arndt TL, Richter RJ, Shih DM, Tward A, Lusis AJ, Jack R.M., Costa L.G., Furlong C.E. 2003. Expression of human paraoxonase (PON1) during development. Pharmacogenetics 13:357-364.

2. Costa LG, Cole TB, Furlong CE. 2003. Polymorphisms of paraoxonase (PON1) and their significance in clinical toxicology of organophosphates. J. Toxicol. Clin. Toxicol. 41:37-45.

3. Friedrich AL, Lee JY, Volitakis I, Cherny R, Cowie TF, Venter DJ, Cole TB, Palmiter R.D., Koh J.Y., Bush A.I. 2003. Synaptic zinc exchange with the perivascular space induces cerebral amyloid angiopathy in an animal model of Alzheimer’s disease. J. Neurosci. (submitted).