Preventing Alzheimers from developing is a goal of Raphael Kopan, Ph.D., professor of molecular biology and pharmacology at the Washington University School of Medicine. The moss plant (Physcomitrella patens) studied in the laboratory of Ralph S. Quatrano, Ph.D., Spencer T. Olin Professor and chair of the WUSTL biology department on the Danforth Campus, might inch Kopan toward that goal. Heres how.
The gene presenilin in mammals provides the catalytic activity for an enzyme called gamma secretase, which cleaves, or cuts, important proteins Notch, Erb4 and the amyloid precursor protein (APP), all key components of communication channels that cells use to arbitrate functions during development. Two genes occur in mammals in which mutations cause an earlier onset of Alzheimers. One is APP, where a fragment of the protein accumulates in amyloid plaques, associated with the disease. Another common site for mutations is found in presenilin (PS) proteins. The enzyme gamma secretase contains PS and works to dispose of proteins stuck in the cellular membrane.
This enzyme with PS at its core mediates two cellular decisions. One is to cut APP and as a byproduct, generate the bad peptide associated with Alzheimers; the other is to cut the Notch protein in response to specific stimuli. Notch is then free to enter the nucleus of cells where it partakes in regulating normal gene expression. Without Notch activity, a mammal has no chance of living.
Notch is a part of short-range mammalian communication channel, and for years it has been known to have a working relationship with PS. However, Notch is absent in plant cells, and presenilin function in plants remained mysterious until Quatranos post-doctoral researcher, Abha Khandelwal, Ph.D., arrived at Washington University and was interested in understanding signal transduction in plants.
When I searched the literature, the plant signal transduction pathways were not very well documented as are the mammalian counterparts such as Notch, said Khandelwal. Meanwhile, my husband Dilip Chandu, Ph.D., was working in the Kopan lab interested in ways to study functions of presenilin without interference from its predominant substrate Notch.
This encouraged Khandelwal to search for the PS gene in the genomes of plants including the recently sequenced moss (Physcomitrella patens) genome, for which the Quatrano lab had access. In addition to the known Arabidopsis presenilin, she found the gene in Physcomitrella and asked, What is PS doing in moss" Is it acting as an enzyme or does it have a different function"
Forming a collaboration
Moss, like yeast, has this great ability where you can actually select a gene and remove it, mutate it, or replace it with another gene from any source. This approach is how we begin to discern a genes value and function in moss, said Quatrano, who was a world leader in getting the moss genome sequenced. It is an excellent system to experimentally discern gene function because of this property as well as others that we and a worldwide consortium have developed over the last several years.
Thus, collaboration was born. By engaging the expertise of the team in the Kopan lab, the Quatrano lab proceeded to start experimenting with PS in moss, which finally resulted in a fruitful combined project, the results of which was recently reported in the Proceedings of the National Academy of Science. Khandelwal proceeded to remove presenilin, and the result was an obvious change- a phenotype. Moss lacking presenilin looked different, growing with straight, rigid filaments instead of curved and bent filaments like the parent moss with the presenilin gene intact.
That showed the gene has an obvious function that obviously, did not require Notch. We just dont know exactly what it is yet, but we have proposed a hypothesis to be tested, Quatrano said.
The phenotype piqued Kopans interest: He saw the potential of looking at the role of presenilin independent of Notch. Khandelwal and Chandu took the phenotype, switched out a mammalian form of presenilin into the phenotype, and rescued it. Similarly, inserting the moss gene in mammalian cells resulted in reversing some of the losses experienced by animal cells lacking presenilin function, testifying that the human and moss proteins had an evolutionary conserved function.
In the moss, they were very nearly interchangeable, Quatrano said. This suggested that presenilin has a role outside the Notch pathway and may provide clues in mammalian systems as to its primary role, independent of its substrate in mammalian cells.
We were amazed to realize that genes from moss and humans were not only structurally conserved but also shared similar functions, Khandelwal said.
Moonlighting protein in mammals
We spent a lot of time trying to find an activity of PS to circumvent cleavage of APP, which has been very difficult, Kopan said. Importantly, the human protein acted in plant cells even if its enzymatic activity was removed by mutation. We stumbled upon an observation that presenilin proteins in mammals can perform other functions besides the enzymatic ones, that is, outside its role as gamma secretase. Were now looking closely to define this moonlighting functions and determine their contribution to disease.
In moss, the mutant phenotypes suggest presenilin might play a role in signal gathering, cytoskeleton organization and/or cell wall composition and organization. Quatrano and Khandelwal are checking that out. Kopan, Chandu and others are searching for presenilins moonlighting activities in mammalian cells.
As a developmental biologist, my job is to translate the genetic code as if it were a manufacturers manual, and that is accomplished by gaining detailed understanding of genes and protein function, Kopan said. Unfortunately, were doing it one gene at a time, slowly building networks, figuring out what the context is. We cant think of all of it at once. We have to look at a small subset of genes and how they work with their friends, and hope that our observations will fit together in one coherent network.
Quatrano said the collaboration between the two labs is a reflection of what the Genomic Age can do.
Today, sitting at your computer, you can data mine genomes from hundreds of microorganisms, animals, fungi, insects and plants, and youre seeing more evidence of genes being conserved in widely different organisms, Quatrano said. This collaboration is a perfect example of bringing two labs together that on the surface have nothing in common other than one protein and two people who were aware of the interests of the other. Its led to a significant contribution that hopefully will lead to further clues as to the function of presenilin.
With this study, the Kopan and Quatrano labs and others could use this outstanding plant model not only to understand some of the off target affects during Alzheimers Disease therapy, but to unravel novel interactions and pathways in plants.
|Contact: Tony Fitzpatrick|
Washington University in St. Louis