Systems Biology Approaches Get Wnt of Progranulin’s Role in FTD

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

Comments

    • User Profile Image Anja Capell
      DZNE-German Center for Neurodegenerative Disease
    • Posted:

    I think this is nice, convincing work. However, lots of new questions occur and remain to be answered.

    Rosen et al. clearly show (with an overwhelming set of data) for the first time that reduced progranulin (GRN) levels in shRNA-expressing human neuronal progenitor cells, in brains from GRN knockout mice, or in brains of FTLD patients with GRN loss-of-function mutations, result in upregulation of activating components of the Wnt signaling pathway, whereas inhibitors of the Wnt pathway are downregulated. The enhanced Wnt signaling due to GRN deficiency was also observed in mature differentiated cells, and did not depend on cell proliferation. This is of particular interest, since in neurodegenerative diseases, adult differentiated neurons are affected. How GRN expression mechanistically affects the expression of components of the Wnt pathway is not addressed by the authors.

    It would be interesting if overexpression of GRN has the opposite effect. In schizophrenia, increased NRG1-, BDNF- and TGF-β signaling and decreased Wnt signaling has been reported (Kalkman, 2009). Since Rosen et al. elegantly show that Wnt signaling is beneficial for neuronal survival upon GRN deficiency, it would be interesting to analyze if growth factor withdrawal in general affects Wnt signaling.

    Moreover, Wnt signaling is only increased in GRN mutation carriers, not in FTLD-TDP cases without GRN mutation; therefore, Wnt seems to be no general hallmark of FTLD-TDP, and whether increased Wnt signaling is beneficial for all FTLD-TDP cases remains to be shown. In other neurodegenerative diseases, such as AD and PD, altered Wnt signaling has been reported. Also, Wnt plays a role in maintenance of survival of neurons as a kind of synaptotropic factor. Therefore, I think it is unlikely that the Wnt signaling is specifically involved in FTLD-TDP.

    Influencing Wnt signaling pathways might provide therapeutic benefits, though it needs to take into account that Wnt also occurs in oncogenic processes. The Wnt pathway would be a negligible diagnostic tool for GRN mutation carriers because GRN expression in any body fluid is the best diagnostic tool. However, variations in Wnt signaling might contribute to the variable age of disease onset within patients carrying the identical GRN mutation. To investigate whether protective Wnt signaling plays a role for the low disease penetrance for GRN mutation carriers might be quite exciting.

    Since we have shown that lysosomal alkalization enhances GRN expression (ARF related news story on Capell et al., 2011), I find it very interesting that the study confirmed that GRN plays an important role in lysosomal function, and that lysosomal genes represent the most significant expression changes in nine-month-old GRN-/- mice.

    References:

    Kalkman HO. Altered growth factor signaling pathways as the basis of aberrant stem cell maturation in schizophrenia. Pharmacol Ther. 2009 Jan;121(1):115-22. PubMed.

    Capell A, Liebscher S, Fellerer K, Brouwers N, Willem M, Lammich S, Gijselinck I, Bittner T, Carlson AM, Sasse F, Kunze B, Steinmetz H, Jansen R, Dormann D, Sleegers K, Cruts M, Herms J, Van Broeckhoven C, Haass C. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J Neurosci. 2011 Feb 2;31(5):1885-94. PubMed.

    View all comments by Anja Capell
    • User Profile Image Jane Wu
      Northwestern University Feinberg School of Medicine
    • Posted:

    The study by Dan Geschwind and colleagues using WGCNA is novel and highly interesting, not only providing a general view of transcriptional alterations associated with reduced granulin (GRN) expression, but also uncovering a previously unknown link between GRN and Wnt pathways. Consistent findings of changes in expression of apoptosis and ubiquitination pathway genes in GRN-knocked down neurons and frontotemporal dementia (FTD) brain tissues suggest the clinical relevance of the results. This elegant work represents one of the first systematic studies of neural transcriptome changes in GRN-deficient FTD cases, and will likely stimulate further research in both mechanistic understanding of FTDs and new therapeutic development.

    View all comments by Jane Wu

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References

News Citations

  1. San Diego: Progranulin, Wnt, and Frizzled, Frazzle Neurons in FTD
  2. Required Reading—InteracTomes for AD, Aging, APP
  3. Progranulin—Curbs Phagocyte Appetites, Protects Neurons?

Paper Citations

  1. Zhang B, Horvath S. A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol. 2005;4:Article17. PubMed.
  2. Miller JA, Oldham MC, Geschwind DH. A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging. J Neurosci. 2008 Feb 6;28(6):1410-20. PubMed.
  3. Miller JA, Horvath S, Geschwind DH. Divergence of human and mouse brain transcriptome highlights Alzheimer disease pathways. Proc Natl Acad Sci U S A. 2010 Jul 13;107(28):12698-703. PubMed.
  4. Chen-Plotkin AS, Geser F, Plotkin JB, Clark CM, Kwong LK, Yuan W, Grossman M, Van Deerlin VM, Trojanowski JQ, Lee VM. Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration. Hum Mol Genet. 2008 May 15;17(10):1349-62. PubMed.
  5. Kao AW, Eisenhut RJ, Martens LH, Nakamura A, Huang A, Bagley JA, Zhou P, de Luis A, Neukomm LJ, Cabello J, Farese RV, Kenyon C. A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells. Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4441-6. PubMed.
  6. Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, Ma X, Ma Y, Iadecola C, Beal MF, Nathan C, Ding A. Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice. J Exp Med. 2010 Jan 18;207(1):117-28. PubMed.
  7. Pickford F, Marcus J, Camargo LM, Xiao Q, Graham D, Mo JR, Burkhardt M, Kulkarni V, Crispino J, Hering H, Hutton M. Progranulin is a chemoattractant for microglia and stimulates their endocytic activity. Am J Pathol. 2011 Jan;178(1):284-95. PubMed.

External Citations

  1. WGCNA
  2. DAVID

Further Reading

Papers

  1. Kao AW, Eisenhut RJ, Martens LH, Nakamura A, Huang A, Bagley JA, Zhou P, de Luis A, Neukomm LJ, Cabello J, Farese RV, Kenyon C. A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells. Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4441-6. PubMed.
  2. Chen-Plotkin AS, Geser F, Plotkin JB, Clark CM, Kwong LK, Yuan W, Grossman M, Van Deerlin VM, Trojanowski JQ, Lee VM. Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration. Hum Mol Genet. 2008 May 15;17(10):1349-62. PubMed.
  3. Pickford F, Marcus J, Camargo LM, Xiao Q, Graham D, Mo JR, Burkhardt M, Kulkarni V, Crispino J, Hering H, Hutton M. Progranulin is a chemoattractant for microglia and stimulates their endocytic activity. Am J Pathol. 2011 Jan;178(1):284-95. PubMed.
  4. Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, Ma X, Ma Y, Iadecola C, Beal MF, Nathan C, Ding A. Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice. J Exp Med. 2010 Jan 18;207(1):117-28. PubMed.
  5. Capell A, Liebscher S, Fellerer K, Brouwers N, Willem M, Lammich S, Gijselinck I, Bittner T, Carlson AM, Sasse F, Kunze B, Steinmetz H, Jansen R, Dormann D, Sleegers K, Cruts M, Herms J, Van Broeckhoven C, Haass C. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J Neurosci. 2011 Feb 2;31(5):1885-94. PubMed.

Primary Papers

  1. Korade Z, Mirnics K. Wnt signaling as a potential therapeutic target for frontotemporal dementia. Neuron. 2011 Sep 22;71(6):955-7. PubMed.
  2. 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. 2011 Sep 22;71(6):1030-42. PubMed.

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