29 September 2007. Just over a year ago, two groups reported their findings that null mutations in the gene for the secreted growth factor progranulin are a frequent cause of frontotemporal dementia, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U, see ARF related news story). Shortly thereafter, other groups identified a major protein that makes up the inclusions found in FTLD-U (and in amyotrophic lateral sclerosis, ALS) as TDP-43, an obscure nuclear protein normally involved in exon splicing (see ARF related news story).
Progranulin and TDP-43 travel in different circles, and since their discovery, one obvious question has been how their paths might cross to cause neurodegeneration. In the September 26 Journal of Neuroscience, Leonard Petrucelli and colleagues from the Mayo Clinic College of Medicine in Jacksonville, Florida, report that decreasing progranulin expression leads to pathological processing of TDP-43 by caspases in cultured cells. Without progranulin, TDP-43 gets cleaved in a caspase-dependent manner, which can lead to translocation from its normal home in the nucleus to the cytosol.
The abnormal processing resembles what happens to TDP-43 in FTLD-U or ALS, the authors show, suggesting that loss of TDP-43 function due to inappropriate cleavage, translocation, or inclusion formation could play an important role in neurodegeneration.
To study the effects of progranulin on TDP-43 processing, first authors Yong-Jie Zhang and Ya-fei Xu used siRNA to knock down progranulin expression in HeLa epithelial cells and in H4 neuroglioma cells. In both cases, as progranulin levels fell, the scientists observed cleavage of TDP-43 into two insoluble fragments. The scientists implicated caspase 3 in the cleavage in several ways: they showed that progranulin knockdown resulted in increased caspase 3 activity in the cells, that caspase inhibitors blocked the proteolysis of TDP-43, and that treating TDP-43 in vitro with caspase 3 or caspase 7 generated similar fragments. The scientists concluded that suppressing progranulin expression is sufficient to trigger the breakdown of TDP-43 through a caspase-dependent mechanism.
The fragments produced in progranulin-deficient cells resembled those found in brains of FTLD-U patients, where TDP-43 relocates from the nucleus to cytoplasmic inclusions. Because of this, the investigators looked further at the consequences of TDP-43 cleavage on protein distribution in their cultured cells. Progranulin knockdown did not noticeably change TDP-43 distribution, whereas staurosporine, a stronger activator of caspase 3, was able to drive TDP-43 protein from its normal location in the nucleus out to the cytosol. That redistribution depended on caspase 3 cleavage, since it was blocked by a mutation in TDP-43 that destroyed a caspase cleavage site.
The effects of staurosporine mimic the change in TDP-43 distribution that occurs in FTLD-U and ALS, where the investigators observe an early loss of nuclear protein and accumulation of cytoplasmic staining, followed by accumulation of fibrillar inclusions in the cytosol.
The results do not show how a reduction in progranulin activity leads to caspase activation and TDP-43 cleavage. In addition, researchers need to determine if the shorter TDP-43 fragments are more fibrillogenic, a property that would explain the formation of inclusions. The intriguing supposition is that loss of progranulin leads to loss of functional TDP-43, which leads to cell death, but that, too, remains to be proven. Nonetheless, the authors envision using their cell culture models as screening tools to find other compounds that specifically affect the processing and redistribution of TDP-43.—Pat McCaffrey.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, Pickering-Brown S, Dickson D, Petrucelli L. Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci. 2007 Sep 26;27(39):10530-4. Abstract