GLOing Reports from Gene Profiling of Mouse Models
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Despite the vastly heterogeneous nature of mammalian brains, researchers are pressing on with gene profiling experiments aimed at identifying potential risk factors for neurodegenerative diseases, including Alzheimer’s disease (AD). Two such studies appeared in last week’s Proceedings of the National Academy of Sciences and Human Molecular Genetics. In PNAS, an international collaboration led by Juergen Goetz from the University of Zurich, reports gene profiling of transgenic mice that express mutated tau (P301L). These animals develop a murine equivalent of frontotemporal dementia with neurofibrillary tangles.
First author Feng Chen and colleagues used Affymetrix DNA chips to compare expression patterns of 6,000 genes in wild-type and tau-mutant animals. Remarkably, statistical analysis of the data revealed that just one gene was differentially expressed, namely that for the enzyme glyoxalase I (GLO). Together with a second enzyme, glyoxalase II, and the cofactor glutathione, glyoxalase I detoxifies α-ketoaldehydes.
To test whether this finding is relevant, the authors used in-situ hybridization to examine expression of GLO directly. They found that the enzyme is expressed throughout the brain, and not just in neurons. Moreover, the enzyme is more highly expressed in tau-mutant mice than in wild-type, the scientists report. Next, the authors turned to samples of postmortem human brain tissue to test if glyoxalase expression is altered in human AD brain, and indeed they found the enzyme to be more highly expressed in people who had had AD than in controls.
As always, when studying postmortem samples, it remains uncertain what the cause-and-effect relationship between GLO and AD might be. The authors did find that, in human neurons, there is an inverse relationship between the GLO enzyme and phosphorylated tau expression, as judged by immunostaining. As GLO is involved in preventing the formation of advanced glycation end products, which can render tau resistant to proteolysis, it may be required for regulation of tau metabolism.
In the second paper, Hemachandra Reddy and colleagues performed a microarray-based gene expression analysis of the Tg2576 strain, a widely used mouse model that overexpresses human mutant AβPP. These scientists, from Oregon Health Sciences University, compared expression of 11,000 genes in wild-type and transgenic animals at two months, five months, and 18 months of age. They found that gene expression differences grew as the animals aged. At two months there were 83 upregulated and 26 downregulated genes in the transgenic animals; by 18 months there were 108 and 149, respectively.
Reddy et al. write that most of these genes were related to mitochondrial energy metabolism. They validated their data by measuring expression changes of select genes with Northern blots. Focusing on three of the genes, ATPase-6, heat shock protein 86, and programmed cell death gene 8, the authors found that in transgenic animals these were overexpressed in pyramidal neurons of the hippocampus and cortex. Dual detection of ATPase-6 and 8-hydroxyguanosine, a marker for oxidative damage, suggested a link between overexpression of the mitochondrial protein and damage due to oxidation.
Evidence is growing for a link between mitochondrial damage, reactive oxygen species, and neurodegeneration. To quote but one recent example, work from Jie Shen’s lab has shown that mutations that cause a rare form of Parkinsonism also result in changes in mitochondrial gene expression. But interestingly, much like Goetz’s group, Shen and colleagues found far fewer genes to be affected, and theirs were mostly downregulated (see ARF related news story).
At this point, a mechanistic interpretation of changes in global brain gene expression patterns remains challenging (see ARF related news story).—Tom Fagan
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Primary Papers
- Reddy PH, McWeeney S, Park BS, Manczak M, Gutala RV, Partovi D, Jung Y, Yau V, Searles R, Mori M, Quinn J. Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Hum Mol Genet. 2004 Jun 15;13(12):1225-40. PubMed.
- Chen F, Wollmer MA, Hoerndli F, Münch G, Kuhla B, Rogaev EI, Tsolaki M, Papassotiropoulos A, Götz J. Role for glyoxalase I in Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 May 18;101(20):7687-92. PubMed.
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Nathan Kline Institute/NYU Langone School of Medicine
In the provocative report by Chen et al., oligonucleotide array analysis was performed using whole brain from P301L-transgenic mice as an input source of RNA. P301L mice express the longest human brain tau isoform, i.e., "big tau," with a pathogenic mutation that in humans results in tauopathy associated with frontotemporal dementia (FTDP-17). Chen et al. use an extremely stringent analysis scheme to identify one gene that they report upon exclusively. The gene, glyoxalase I (GLO), encodes an enzyme that detoxifies carbonyls and reduces the formation of advanced glycation end products (AGEs), which are found in abundance in Alzheimer’s disease (AD) brains and related neurodegenerative disorders. An interesting facet of this study is that the group used microarray analysis to identify GLO as the sole target for their publication. Typically, microarray analysis is employed for high-throughput analysis to identify dozens to hundreds of transcripts. Instead, Chen et al. chose to whittle down potential targets from an original pool of 133 upregulated genes and 99 downregulated genes (relative to non-transgenic mice). When statistical analyses were combined with signal intensity filters, 34 upregulated and 12-down regulated genes remained. Stringent pair-wise analysis detected only GLO, which was determined to be significantly up regulated 1.6 fold in P301L mice versus controls. The report marks an interesting paradigm shift from conventional microarray analysis and provided a provocative target in a mouse model of tauopathy, which was further validated by a myriad of molecular and immunocytochemical based technologies.
In a related paradigm, Reddy et al. used microarray analysis, in this case spotted cDNA microarrays, in a well-established mouse model of cerebral amyloidosis, the Tg2576 APP transgenic mouse, at three time points (two months, five months, both pre-amyloid deposition, and 18 months, when extensive amyloid has deposited). Employing a conventional array analysis scheme, 83 upregulated and 26 downregulated genes were observed relative to control mice at two months, 54 upregulated and 30 downregulated genes at five months, and 108 upregulated and 149 downregulated genes were observed at 18 months. Selected genes were validated using Northern blot analysis, in situ hybridization, and immunocytochemistry. Two classes of transcripts—mitochondrial genes and apoptotic genes—were consistently upregulated at all three time points, implicating these genes in the formation of cerebral amyloidosis and demonstrating their relevance towards understanding mechanisms of AD pathology.
Taken together, these two reports illustrate the utility and flexibility of expression profiling paradigms in animal models of tauopathy and amyloid deposition, respectively. The statistical mode of detection and experimental analysis of genes can range from individual mRNAs to large classes of transcripts, depending upon the experimental design and biostatistical parameters. Notably, both studies used relatively large tissue dissections (e.g., whole brain and cortex) as input sources of RNA. It is interesting to speculate whether GLO and/or additional genes would be identified if the input sources were microdissected vulnerable regions (e.g., hippocampus) or individual cell types prone to neurodegeneration.
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