Ezra Rosen, a student in the laboratory of Dan Geschwind, and independent researcher Eric Wexler, all at the University of California in Los Angeles, presented a pair of posters detailing the PGRN-Wnt connection. Premature stop codons in the granulin gene (GRN) that encodes progranulin are a common cause of frontotemporal dementia (FTD). In a chromosomal coincidence, GRN sits just a megabase away from tau, another FTD-related gene on chromosome 17 (see ARF related news story on Cruts et al., 2006 and Baker et al., 2006). Progranulin’s role in neurons is unknown. “Our very simplistic hypothesis is that progranulin is some sort of trophic factor,” Wexler told ARF in an interview. Unique among the dementias, Wexler said, PGRN-associated FTD appears to be caused by a haploinsufficiency, with one valid GRN gene still present. Yet in the brains of people who had FTD, PGRN expression was actually higher than normal, Wexler said, mostly due to PGRN in microglia (see also Philips et al., 2010). “It does not correlate,” he said. “If the microglia can make it from the gene that is still good, why can’t the neurons?”

A clue turned up through Wexler’s separate interest in Wnt signaling, which is essential throughout the body, with roles in neurogenesis (see ARF related news story on Lie et al., 2005) as well as in dementia (see ARF related news story on De Ferrari et al., 2007). As outlined in one poster, Wexler and Rosen started with a study of Wnt1 signaling in fetal human neural progenitors (hNPs) that they differentiated into neurons. After stimulating the cells with Wnt1, the researchers used an unbiased expression screen to examine changes to the transcriptome over periods ranging from two hours to three days. They found widespread alterations in RNA levels of genes related to cell death processes and to neurodegeneration, including both GRN and presenilin-1, which rose and fell at different time points.

The researchers were particularly interested in the Wnt-PGRN connection. They confirmed, via Western blotting, that Wnt1 reduced PGRN protein levels in the hNP-derived neurons. They discovered that knocking down PGRN by half, via RNA interference, increased Wnt1 expression by at least twofold in the hNP-made neurons. The result is a feedback loop in which ever-increasing Wnt1 levels repress PGRN expression more and more, leading to even greater induction of Wnt1. This, the authors suggest, is one possible reason why progranulin might be down in the neurons of people with FTD, even when they have one good GRN gene. Microglia, which make abundant progranulin, may not rely on this pathway to regulate GRN expression. The researchers confirmed their results in postmortem tissue from people who had GRN-mediated FTD; in this tissue they found upregulation of Wnt signaling-related genes compared to control tissues.

In their second poster, Rosen and colleagues described hunting for downstream consequences of progranulin deficiency that might help explain its role in dementia. They infected the hNP-derived neurons with a lentiviral construct carrying a doxycycline-inducible GRN RNAi. Cells treated with the inducer had less than 10 percent of the normal levels of progranulin and altered expression of 153 other genes, compared to untreated cells. Using a standard gene ontology database, the researchers determined related gene groups, or modules, that were most affected by progranulin knockdown. “The only disease category that is represented is dementia, and the only signaling pathway that is represented is Wnt signaling,” Wexler said. Among the dementia-linked genes with altered expression were glycogen synthase kinase 3β (GSK-3β) protein phosphatase 2 A (PP2A), and APC, which are scaffolds mediating presenilin-1/β-catenin interactions. Other affected genes were Wnt1, Frizzled-2, and other signaling and pro-apoptotic genes.

Thus far, the researchers knew that Wnt1, Frizzled-2, and PGRN were all involved together in lab-grown neurons. Next, they sought to confirm their finding in human and animal studies. They compared their GRN-influenced gene set to data from a previously published study on gene expression in brain samples from people who had FTD (Chen-Plotkin et al., 2008). Between the two datasets, a handful of genes overlapped. Frizzled-2 was one of the most highly expressed in both the human and cell studies, so the researchers analyzed this gene further.

For an animal model, coauthor Robert Farese of the University of California in San Francisco provided an as-yet unpublished GRN knockout mouse. These animals show microglial activation by six months and neural loss by 18 months of age, but they had elevated Frizzled-2 in the neocortex at six weeks, suggesting Frizzled-2 upregulation is an early response to GRN deficiency. Frizzled-2 upregulation might be a contributor to neurodegeneration, or might be an insufficient attempt to compensate for GRN loss, the scientists posited. They found that Frizzled-2 knockdown in GRN-deficient hNP-derived neurons led to an increase in cell death, suggesting it could be a neural protector of some kind.

Frizzled-2 upregulation might be a useful early marker for FTD, Wexler speculated. The researchers also looked at other dementia model mice, he told ARF, but only saw upregulation of Frizzled-2 in GRN knockout animals. “It seems to be highly related to this disease,” he said. Indeed, gene association studies have not implicated Frizzled-2 in any other major neurodegenerative disease.

Interestingly, the Frizzled-2 gene is one of a handful of genes that sit between the tau and GRN genes on chromosome 17. “This does seem like a real coincidence,” Wexler said. He hypothesizes that non-coding RNAs in the same region might somehow affect these neighboring genes in a manner that leads to disease, although he admits that is “hand waving” at this point. To address this hypothesis, Wexler is collecting tissue samples from people with GRN-mediated FTD and their unaffected siblings. He plans to make pluripotent stem cells, then neurons, from these samples and prepare RNA libraries. Then, he intends to sequence the RNA to look for chromosome 17 patterns that are common among FTD cells.

“The identification of the interaction between progranulin and Wnt signaling is important and requires further study,” wrote Philip Van Damme of VIB Leuven, Belgium, who also studies GRN-mediated neurodegeneration, in an e-mail to ARF. “An important issue will be to what extent Wnt signaling mediates the functional consequences of progranulin deficiency. Is it a secondary effect or integral part of the detrimental effects of progranulin deficiency?”

In sum, the research points to a role for Wnt1 in silencing GRN in FTD neurons, potentially aided and abetted by Frizzled-2. The Wnt-GRN pathway is thus a potential therapeutic target, but the widely interconnected signaling networks could complicate the search for a therapy, Wexler suggested. “This is as much a disease of dysregulation as of anything else,” he said. “It may not be as easy as giving people more progranulin.”—Amber Dance.