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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests The Division of Quantitative Sciences includes the laboratories of Analytical, Cellular, and Molecular Microscopy (ACMM), Microarray Technology, Computational Biology, Molecular Epidemiology, and Mass Spectrometry and Proteomics. The Division’s laboratories use objective measures to define pathophysiologic events and processes. The ACMM laboratory prepares samples by either paraffin or frozen methods and 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 they enable researchers to determine where in a cell particular molecules are located. An archive of pathology tissues in paraffin blocks (Van Andel Tissue Repository, or VATR) is being accumulated with the cooperation of local hospitals. The archive currently has approximately 250,000 paraffin blocks representing 150,000 cases. In collaboration with Tom Barney from VAI-IT, clinical data is being added into VATR for hundreds of the samples each week by digital parsing of pathology report texts sent electronically from the hospital files. VATR is used to track samples coming from the hospitals, along with all of the data and images generated from research. Images from the Aperio ScanScope are automatically imported into VATR and associated with the appropriate sample. The ACMM 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 arrays, miRNA arrays, and array CGH using the Agilent microarray platform and cDNA platform capability. Samples can be prepared from a variety of species. Genomic DNA or total RNA from a wide range of tissues including blood and fresh or frozen tissues have been analyzed. A recent gene expression discovery was made using archived newborn blood spots, in collaboration with Dr. Nigel Paneth at MSU. We showed that thousands of gene signatures can be obtained using low-resolution gene expression arrays, enabling clinical research into the origins, epidemiology, and diagnosis of human pediatric diseases. Feature extraction software reads and processes the raw microarray image files in an automated mode. Application-specific QC reports summarize the results and provide an accurate quality assessment. The output files are compatible with statistical analysis packages such as R and GeneSpring. Microarray technology plays an important part in the discovery of genetic signatures, copy number variations, and biomarkers for therapeutic purposes. Hauenstein Parkinson’s Center Throughout 2008 we continued our collaboration with the Hauenstein Parkinson’s Center, collecting blood samples and control samples from 216 individuals. Mutations/polymorphisms in the NR4A2 gene are being studied by DNA sequence analysis, motivated by our previous identification of this gene in a genomic screen for neuroprotective factors. We are particularly interested in polymorphisms in the DNA-binding domain of NR4A2, as changes to this region of the gene are most likely to affect its function. Identification of novel Parkinson-modifying genes with siRNA screening Small interfering RNA (siRNA) technology allows the specific knockdown of any mRNA/protein pair. Under the direction of VARI’s Jeff MacKeigan, postdoctoral fellow Brendan Looyenga has begun to use a subset of the siRNA library developed by Qiagen to individually target several classes of enzymes having pharmaceutical potential. Specifically, we are continuing a project to identify molecules that attenuate oxidative stress–induced toxicity in dopaminergic neurons; our initial focus is on phosphatases and kinases. We are validating the initial screening studies and we hope to extend these studies to include nuclear hormone receptors and G protein–coupled receptors. 40

VARI | 2009 Mouse models of Parkinson disease James Resau and Brendan Looyenga, in collaboration with VARI’s Bart Williams, are generating novel rodent models of dopaminergic cell loss in the brain using the DAT-cre mouse line, which specifically expresses the cre recombinase in dopaminergic neurons of the brain. Projects based on the DAT-cre mouse model include the following. • Imaging and isolation of primary dopaminergic neurons from mouse brain. Brendan Looyenga is continuing a project to generate mice that specifically express the LacZ reporter gene in dopaminergic neurons. 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. The DAT-cre/ ROSA26 mice will also provide a source of highly pure dopaminergic neurons for in vitro studies. • Functional roles of the phosphatase PTEN in dopaminergic neurons. The phosphatase PTEN is a crucial signaling node in mammalian cells. PTEN catalyzes the removal of 3´ phosphates from phosphoinositol (PI), effectively antagonizing the activity of PI3-kinase. Loss of PTEN results in constitutively elevated levels of the phospholipids PI(3,4)P 2 and PI(3,4,5)P 3 , which strongly activate downstream effectors including AKT/PKB and mTOR. Hyperactive AKT is traditionally associated with cell survival and proliferation, while hyperactivation of mTOR is associated with cellular hypertrophy. Interestingly, these two effects often occur in mutually exclusive fashion depending on the status of the cell in which PTEN is deleted. Terminally differentiated cells usually display hypertrophy, and they rarely reenter the cell cycle upon PTEN deletion. To confirm that the DAT-cre mice only express the cre recombinase in terminally differentiated cells, we have crossed them to mice bearing a conditional knock-out allele for PTEN (PTEN loxP ). As expected, dopaminergic neurons in DAT-cre/PTEN loxP mice demonstrate constitutive activation of AKT and mTOR; however, they fail to develop neuronal tumors, suggesting that the loss of PTEN in dopaminergic neurons does not induce hyperproliferation. These cells do appear larger, consistent with the induction of hypertrophy. We are currently quantifying these observations for publication. We are also using the DAT-cre/PTEN loxP mice to elucidate the connection between PTEN and mitochondrial function. This connection is based on the ability of PTEN to increase expression of the familial Parkinson gene, PINK1, in cultured tumor cells. Several studies have shown that PINK1 plays a crucial role in the maintenance of mitochondrial fission/fusion homeostasis, implying that PTEN indirectly regulates mitochondrial function by controlling cellular PINK1 levels. To determine whether PTEN regulates PINK1 and mitochondrial function in normal cells, we are analyzing PINK1 expression levels and mitochondrial function in the dopaminergic neurons of DAT-cre/PTEN loxP mice. We hypothesize that loss of PTEN in these cells will result in decreased PINK1 expression, imbalances in mitochondrial fission/fusion, and increased oxidative stress. Studies in brain tissues and cultured primary neurons are ongoing. Other highlights Our GRAPCEP mentorship program continues for the ninth year and is now funded by Schering Plough. In 2008 we had two students from GRAPCEP, several undergraduate summer interns, and a graduate school student rotation. 41

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