Research Interests
The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), the Laboratory of Microarray Technology, the Laboratory of Computational Biology, the Laboratory of Molecular Epidemiology, and the Laboratory of Mass Spectrometry and Proteomics. The Division’s laboratories use objective measures to define pathophysiologic events and processes.
The ACMM laboratory has programs in pathology, histology, and imaging to describe and visualize changes in cell, tissue, or organ structure. Our imaging instruments allow us to visualize cells and their components with striking clarity, and enable researchers to determine where in a cell specific molecules are located. An archive of pathology tissues in paraffin blocks (Van Andel Tissue Repository) is being accumulated with the cooperation of local hospitals, and the data on the samples is being converted to computerized files in collaboration with Tom Barney from VAI-IT. The lab also carries out research that will improve our ability to quantify images. We are able to image using either fluorescent (e.g., FITC, GFP) or chromatic agents (e.g., DAB, H&E) and separate the components using our confocal, Nuance, or Maestro instruments.
The Laboratory of Microarray Technology provides gene expression analysis using Agilent commercially prepared arrays as well as “home-brewed” cDNA microarrays. In 2007 we produced and used 305 cDNA microarrays and 150 custom protein microarrays. We also used 107 Agilent arrays to genomically characterize a variety of tissues and samples, including archived human blood samples from newborns.
Hauenstein Parkinson’s Center
Throughout 2007 we continued our collaboration with the Hauenstein Parkinson’s Center, collecting blood samples and controls from 154 individuals. Mutations in the parkin gene in a set of families with more than one generation affected by Parkinson disease are being studied by DNA sequence analysis and will be correlated to gene expression data from microarray analysis.
Identification of Novel Parkinson-Modifying Genes with siRNA Screening
Small interfering RNA (siRNA) technology allows the specific knockdown of any mRNA/protein pair. Combined with information from the human genome, this technology has given rise to libraries of siRNAs targeted to every known or predicted gene in the genome. Under the direction of VARI’s Jeff MacKeigan, postdoctoral fellow Brendon Looyenga has begun to use a subset of the siRNA library developed by Qiagen to individually target several classes of enzymes having pharmaceutical potential. We are searching for genes involved in Parkinson disease that may be drug targets for rationally designed therapies.
We are attempting to identify molecules that attenuate oxidative stress–induced toxicity in dopaminergic neurons; our initial focus is on phosphatases and kinases. To date we have screened all of the phosphatases in the human genome and have identified several potential candidates that regulate neuronal cell death in response to 6-hydroxydopamine, a toxic compound used to induce oxidative stress in Parkinson research. We are validating these initial screening studies and are planning the assays required to screen all kinases in the human genome as well. We hope to extend these studies to include nuclear hormone receptors and G protein–coupled receptors.
Mouse Models of Parkinson Disease
James Resau and Brendan Looyenga are generating novel rodent models of dopaminergic cell loss in the brain in collaboration with VARI’s Bart Williams. A key tool for these studies is the transgenic dopamine-transporter/cre (DAT-cre) mouse line, which specifically expresses the cre recombinase in dopaminergic neurons of the brain. The DAT-cre mice will allow us to address the response of such neurons to specific gene deletions and additions; projects based on the DAT-cre mouse model include the following.
- Imaging and isolation of primary dopaminergic neurons from mouse brain. Brendan Looyenga has performed a genetic cross between the DAT-cre strain and ROSA26 reporter strain to generate mice that specifically express the LacZ reporter gene in dopaminergic neurons. The DAT-cre/ROSA26 mice will permit us to visualize and quantify live dopaminergic neurons in vivo. With these mice we will assess the effect of cytotoxic agents (e.g., MMTP, rotenone, or 6-hydroxydopamine) on the number of dopaminergic cells, and more importantly, assess the ability of mice to recover from these insults. These studies will provide insight into the regenerative capacity of the brain when dopaminergic neurons are lost or injured. The DAT-cre/ROSA26 mice will also provide a source of highly pure dopaminergic neurons for in vitro studies.
- Dopaminergic cell regeneration as a function of age. The relationship between age and the likelihood of developing Parkinson disease is well established, though the causal nature of this relationship is unclear. One hypothesis is that the capacity of the brain to regenerate damaged neurons decreases with age, consistent with a gradual loss of brain stem cells that give rise to new dopaminergic neurons. To test this hypothesis in a mammalian system, we will cross DAT-cre and puDTK mice, the latter specifically expressing herpes simplex virus thymidine kinase (hsvTK) in cells that contain cre recombinase. Cells expressing hsvTK are sensitive to ganciglovir (G418) and undergo programmed cell death after systemic treatment. Using the DAT-cre/ puDTK model, we will eliminate dopaminergic neurons at various ages and assess the regenerative potential of these mice. These studies will provide information about the value of therapies intended to stimulate the endogenous regenerative capacity of the brain in Parkinson disease patients.
- Effect of hypoxia-inducible factor signaling on dopaminergic cell survival. Dopaminergic neurons are exquisitely sensitive to oxidative stress (reactive oxygen species), which can lead to cell death by direct mechanisms, such as damage to important cellular biomolecules, and indirect ones, such as the induction of cell death pathways. The latter mechanism may be offset by cell survival pathways, which increase the threshold signal intensity required to induce cell death. Because Parkinson disease is characterized by increased oxidative stress in dopaminergic neurons, therapies that increase cell survival pathways in these neurons may be broadly applicable to decrease cell death in patients.
Other Highlights
Our GRAPCEP mentorship program continues for an eighth year and is now funded by Schering Plough. This year we had three students from GRAPCEP. Dr. Resau is a member of the graduate school committee that established the VAEI Graduate School, which will increase our research and educational opportunities. Also in 2007, Jim Resau had an image selected as one of the Nikon Small World top 100 images, and Bree Berghuis, working with Carrie Graveel, had an image of cMet staining selected for the June 2008 Ventana Calendar.
