26 September 2011. As much as they can enlighten, microarray analyses also overwhelm—drowning researchers with data on hundreds, sometimes thousands, of genes that potentially contribute to a given disease. Newer approaches that emphasize functional networks instead of individual genes have surfaced, and a paper in the September 22 Neuron shows how these methods, in combination with mouse and human gene expression analyses, convincingly link the Wnt signaling pathway to frontotemporal dementia (FTD) caused by progranulin (GRN) haploinsufficiency. Using systems biology, researchers led by Daniel Geschwind at the University of California, Los Angeles, found that GRN-deficient cells express higher levels of genes that typically activate Wnt signaling and lower amounts of genes that dampen this pathway. The work “provides the impetus for further in-depth exploration of Wnt signaling in FTD, and suggests the potential use of Wnt agonists to assuage the neurodegenerative phenotype of [FTD caused by GRN mutations],” the authors write. Some of this work was presented at the 2010 Society for Neuroscience annual meeting in San Diego (see ARF related conference story).
UCLA biostatisticians developed a method that simplifies messy microarray data by arranging genes into functional networks based on their expression patterns in specific tissues (Zhang and Horvath, 2005). Geschwind’s lab used this approach, called weighted gene coexpression network analysis (WGCNA), to identify genes and gene networks that may contribute to normal aging and to AD (see ARF related news story on Miller et al., 2008), as well as to determine how the pathways converge across mice and people (Miller at al., 2010).
In the present study, first author Ezra Rosen and colleagues turned their attention toward FTD, in particular, the 5 to 10 percent of cases due to GRN mutations that cause haploinsufficiency. To get a handle on the pathways that mediate the effects of GRN deficiency, the researchers generated an in-vitro model using short-hairpin RNAs (shRNAs) to reduce GRN expression in primary human neural stem cells. GRN transcript levels dropped 60-74 percent in the knockdown cells, comparable to what is seen in FTD patients. Standard microarray analysis revealed a slew of cell cycle and ubiquitination genes that were enriched in GRN-deficient cells. Consistent with neurodegeneration being the ultimate consequence of GRN insufficiency in people, GRN-inhibited cell cultures also showed more pyknotic, or shrunken nuclei, stronger immunostaining for activated caspase 3, and fewer surviving cells relative to cultures treated with scrambled control shRNA.
To refine and organize the 153 genes associated with GRN loss in the human neuronal stem cell model, the researchers used a gene ontology bioinformatics tool called DAVID. The cell death and apoptosis category came up strong, confirming the cell culture data. Notably, many of the differentially expressed genes, i.e., CD24, WNT1, SFRP1, NKD2, and the Wnt receptor FZD2, are members of the Wnt signaling cascade, with activators of the pathway (WNT1, APC2, FZD2) upregulated and inhibitors (GSK3B, SFRP1, NKD2, CER1) downregulated in GRN-inactivated cells. The scientists confirmed these genes using quantitative RT-PCR and a reporter system that measures Wnt activity.
As further validation, Geschwind’s team used WGCNA along with the gene ontology tool to survey recently published postmortem microarray data from FTD patients, including some with GRN mutations (i.e., GRN+ FTD) and matched controls (Chen-Plotkin et al., 2008). On the whole, the findings held in these human subjects, as well as in gene expression analyses from the brains of young GRN knockout mice. The Wnt receptor FZD2 came up as one of the most consistently upregulated targets in six-week-old GRN-deficient mice, which lack obvious neuropathology or neurodegeneration.
“The overall results prove, beyond any doubt, that the GRN+ FTD pathology is at least in part mediated through dysregulation of the Wnt signaling pathway, and that these changes are in place before the onset of neurodegenerative changes,” noted Zeljka Korade and Karoly Mirnics of Vanderbilt University, Nashville, Tennessee, in a Neuron commentary on the current study.
These scientists, and others, praised the work for its innovative and powerful combination of research tools. They also noted a number of unaddressed issues—among them, how exactly GRN regulates the Wnt pathway, and whether Wnt signaling plays a role in FTD cases not caused by GRN mutations (see Anja Capell's comment below).
Furthermore, Korade and Mirnics ask, What are the compensatory mechanisms that keep the effects of GRN deficiency, presumably present since before birth in affected individuals, in check for some 60 to 70 years, and how do those effects burn out by late adulthood?
And what about glia, where GRN seems to play a key role in suppressing apoptosis (see ARF related news story on Kao et al, 2011; Yin et al., 2010; Pickford et al., 2011)? While the present results “argue for a strong neuronal pathology in response to reduced GRN levels, early contribution of glial dysfunction to the FTD pathology cannot be excluded,” Korade and Mirnics write.
The current findings suggest that targeting the Wnt pathway could hold promise therapeutically—not only for GRN+ FTD, but possibly Alzheimer’s and Parkinson’s diseases, where changes in this signaling cascade have also been reported. Researchers must take caution, though, because Wnt contributes to oncogenic processes. In the meantime, the findings “highlight the most important, missing knowledge” and should “indicate a clear path to the most intriguing future experiments,” Korade and Mirnics wrote.—Esther Landhuis.
Rosen EY, Wexler EM, Versano R, Coppola G, Gao F, Winden KD, Oldham MC, Martens LH, Zhou P, Farese RV, Geschwind DH. Functional Genomic Analyses Identify Pathways Dysregulated by Progranulin Deficiency, Implicating Wnt Signaling. Neuron. 22 Sep 2011;71:1030-1042. Abstract
Korade Z and Mirnics K. Wnt Signaling as a Potential Therapeutic Target for Frontotemporal Dementia. Neuron. 22 Sep 2011;71:955-957. Abstract