Prof. Katheleen Gardiner
The use of reverse phase protein array (RPPA) and Western blot on heat stabilized tissue
Professor Katheleen Gardiner has had an affiliation with the University of Colorado for 20 years where she is engaged in Down syndrome research. Katheleen obtained an undergraduate degree in physics, moved on to study RNA processing in bacteria and then switched focus to study the biology of mammalian systems during her post doc. Since then Katheleen focused her research on understanding the mechanisms of Down syndrome (DS)* on a molecular level. Initially, she was intrigued by the correlation between DS and Alzheimer´s disease (AD). A majority of persons with DS will develop the neuropathology of AD in their late twenties and many will go on to develop an AD-like dementia by 40-50 years of age. Currently, her research focuses on identifying the molecular abnormalities that underlie the learning and memory deficits in DS.
Katheleen is heading a group of scientists in the division of Genetics in the Department of Pediatrics. The department has over 400 faculty members, a majority of clinicians and around 50 basic scientists. Basic, clinical and translational research is concentrated on genetics and biomedical science relevant to pediatric health. Katheleen is also a member of the Linda Crnic Institute for Down syndrome and the Intellectual and Developmental Disabilities Research Center within the Department of Pediatrics.
Direction within Down syndrome research
Down syndrome has long been regarded by many in both basic - and clinical research as too complex a challenge for effective pharmacological intervention. This attitude, however, is changing as a result of demonstrations by laboratories around the world that several different compounds and potential drugs can rescue learning - and memory deficiencies in a major mouse model of DS. The challenges in DS have not become simpler, rather the knowledge of human genes, their genetics, functions, interactions and biochemistry, has dramatically improved over the last few years.
A major focus in DS research is on pre-clinical evaluation of drug treatments for cognitive deficits and there is a push to find drugs that have already been approved for other disease paradigms and to re-purpose them for DS. There are a few clinical trials already on-going and Katheleen is optimistic that, in the future, “we will be able to treat cognitive deficiencies in DS successfully, not reverse everything but drastically improve the cognitive functions”.
Challenges with protein degradation in mouse models in Down syndrome research
Katheleen´s research group is focusing their efforts on the analysis of protein expression and pathways in mouse model systems of DS. This provides crucial insights into the normal functions of specific genes, how these are altered in disease and how they contribute to a disease process, as well as information on drug action, efficacy and side effects.
One of the major challenges when studying proteins and their pathways in the mouse brain is obtaining reproducible and accurate measurement of molecular events. Protein profiles are affected by post-mortem changes in the tissue which begins as soon as the tissue is excised and involve rapid changes in levels of neuropeptides and post-translational modifications, e.g. phosphorylations and acetylations. Depending on how the tissue sample is handled, you may not look at the in vivo profile, which obviously is what scientists strive for. Katheleen has evaluated various techniques to prevent post-mortem changes e.g. protein degradation, and at a conference on DS organized by herself in 2007, she came in contact with the heat stabilization technique for the first time. She evaluated the technology´s performance and has now incorporated heat stabilization into the standard protocol when studying protein profiles in tissue samples, with the exception of when subcellular fractionation is required. Heat stabilization has drastically improved the consistency and reproducibility in their protein profile studies and Katheleen adds “I believe that people need to think more carefully about what exactly they are looking at when they study protein profiles in brain or any other tissue”.
The Stabilizor system is frequently used, for both fresh and frozen samples. For example, the group heat stabilized a great number of brain samples that had been stored in the freezer for over 5 years, dissected out specific brain regions, extracted the proteins and analyzed them by Western Blots and Reversed Phase Protein Arrays. They are very satisfied with the highly reproducible results and Katheleen says “the Stabilizor system is ideal for all tissue samples that you have stored in the freezer, as you really do preserve the protein profile from whatever state the tissue was in when you put it in the system - you preserve it”.
Before implementing heat stabilization the quality of protein profiles depended on who did the dissection. Katheleen explains “I could clearly tell when a less experienced person had done the dissections; the phosphorylation levels were all over the place and, by looking at the protein profiles I could tell that dissection had taken long time”. The Stabilizor system allows students learn how to dissect brains without worrying about wasting precious samples, as the tissue sample remains stable throughout the workflow.
High quality data
As part of their standard tissue workflow in protein profile studies, Katheleen finds the Stabilizor system very easy to use, appreciates the small footprint and concludes that she has a healthy working relationship with Denator. The Stabilizor system has enabled her research group to drastically improve the consistency of sample quality obtained from mouse models, enabling reproducible and consistent protein profile studies. This is advantageous in their pursuit of drugs with the potential for significant benefits to individuals with Down syndrome.
* Down syndrome, also known as trisomy 21, is a chromosomal condition caused by an extra copy of all or part of chromosome 21, thus patients have an extra copy of a large number of genes. Down syndrome is the most common chromosome abnormality in humans and is associated with deficits in cognitive ability, physical growth and characteristic facial features.
Learn more about using mouse to model neurobiological and behavioral aspects of Down syndrome and assess pharmacotherapeutics.
Publications from Prof. Katheleen Gardiner's research group utilizing the Stabilizor technology
Protein profiles in Tc1 mice implicate novel pathway perturbations in the Down syndrome brain. Ahmed et al., Human Molecular Genetics. January 2013.
Keywords: Down syndrome, mouse model, phospho-proteins, RPPA
Expression of trisomic proteins in Down syndrome model systems. Spellman et al., Gene. October 2012. In press, accepted manuscript
Keywords: Down syndrome, quantitative Western Blot, mouse brain
Loss of correlations among proteins in brains of the Ts65Dn mouse model of Down syndrome. Ahmed et al., J. of Proteome research. 2012, 11 (2), pp 1251–1263
Keywords: Down syndrome, Trisomy 21, MAP kinase, Reverse Phase Protein Array (RPPA), phospho-proteins
Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization, M Ahmed et al, Journal of Neuroscience Methods. 2011 Mar 15;196(1):99-106
Keywords: Mouse brain, Western Blot, phospho-proteins