Mutations in the progranulin gene cause a form of frontotemporal dementia, most likely because levels of the protein fall in the central nervous system. How this affects brain function is unknown. Theories pegged progranulin functions as a growth factor or an antiinflammatory protein. More recent work put progranulin front and center in lysosome function, but did not explain what it did there. Now, a trio of studies promises to change just about everything scientists think about this protein, particularly its role in the cell.
In the August 9 eNeuro, researchers led by Thomas Kukar, Emory University in Atlanta, report that, in multiple cell types, progranulin rapidly traffics to lysosomes, where it is cleaved into small, stable granulins. Scientists have known for years that granulins can be generated extracellularly, but this is the first time they have been reported to be produced in lysosomes. After they are made, the granulins remain in place, suggesting they contribute to lysosomal function. The researchers hypothesize that dearth of granulins, rather than progranulin, causes FTD. Levels of both are lower in brain tissue from patients with progranulin mutations compared to controls.
Two additional reports, one from Leonard Petrucelli’s lab at Mayo Clinic, Jacksonville, Florida, and the other from Fenghua Hu, Cornell University, Ithaca, New York, identify cathepsin L, a lysosomal cysteine protease, as a major progranulin processing enzyme. Petrucelli’s work was published in the July 25 Molecular Neurodegeneration; Hu’s paper appeared online in the same journal on August 23. Taken together, the studies provide circumstantial yet compelling evidence that granulins represent the business end of progranulin in lysosomes, say the researchers.
“The aggregate of these papers is incredibly valuable to the progranulin field and for understanding the etiology of FTD,” said Michael Ward of the National Institute of Neurological Disorders and Stroke in Bethesda, Maryland. To top that off, a paper from Morgen Sheng’s lab at Genentech, South San Francisco, California, reveals a role for progranulin in autophagy, a key cell pathway for clearing large cargoes like spent organelles and invading bacteria. The work, published August 4 in the Journal of Experimental Medicine, indicates a widespread negative impact of progranulin loss on cell degradation pathways.
The Sheng report adds to the rapid accumulation of new insights about the cellular functions of progranulins, Ward told Alzforum. “It’s been quite a week for the field,” he said.
In focusing on progranulin’s role as a secreted growth factor, scientists have, for the most part, overlooked its “pro” aspect. Progranlin sports seven and a half cysteine-rich repeats joined by linker regions that can be cleaved to release seven different granulins. Granulins were discovered decades ago in white blood cells, and the prevailing view is that they function as proinflammatory mediators (Bateman et al., 1990). However, they have received little attention of late, mainly due to the lack of specific antibodies to detect them.
In Kukar’s lab, first author Christopher Holler started by meticulously characterizing a dozen commercially available or in-house progranulin antibodies, finding several that recognized one or more fully processed human granulins. To look at cellular processing, Holler developed a novel assay, where he pulsed progranulin-lacking HAP-1 human cancer cells with recombinant progranulin for 24 hours, then chased with plain media. He followed the protein’s fate by western blotting or immunofluorescence microscopy. He found that cells rapidly took up the extracellular progranulin and processed it to granulins. In the chase phase, full-length progranulin disappeared rapidly, sinking to undetectable levels within six hours. In contrast, the granulins persisted, and were still apparent 16 hours later.
Where were the granulins? In HeLa cells fractionated by gradient density centrifugation, Holler found granulins restricted to lysosomes, whereas progranulin was found in other fractions as well, including ER, Golgi, and endosome fractions. High-resolution microscopy in HAP1 cells pinned the proteins to the inner leaflet of LAMP-1 positive lysosomes.
To determine which intracellular proteases process progranulin, Holler treated cells with a panel of inhibitors. Only cysteine protease blockers reduced granulin production. He found that the lysosomal cysteine protease cathepsin L could process progranulin to granulins in vitro, and a specific cathepsin L inhibitor decreased granulin production in HAP1 cells. General lysosome inhibitors also blocked processing: cholorquine, bafilomycin, or concanamycin A previously had been shown to increase progranulin levels in cells, presumably by halting its breakdown; the investigators here showed those same treatments significantly decreased granulin levels in HAP1 cells.
Those results echo the work from Petrucelli and colleagues, who also identified cathepsin L as a progranulin protease. When first author Chris Lee, now at the Biomedical Institute of New Jersey in Cedar Knolls, overexpressed progranulin along with cathepsins B, L, or D in human HEK293 cells, only L caused the parent progranulin to disappear. He found the same in vitro: only cathepsin L, but not B or D, could robustly digest progranulin in a test tube. The investigators mapped the cleavage sites, and found all were in the linker regions between the granulin repeats. This suggests that cathepsin L’s does not function merely to rid cells of progranulin, but instead to process it in an orderly fashion to its component granulins. Some pathogenic progranulin mutations sit close to or overlap with these cleavage sites, suggesting that they might affect processing in lysosomes, Lee told Alzforum. The big challenge now is to show that granulins function in lysosomes, and what they do there, he said.
In the third study, first author Xiaolai Zhou in Hu’s group examined progranulin processing in mouse tissues. They raised an anti-mouse progranulin antibody that picked up full-length progranulin and peptides corresponding to low molecular weight granulins in microglia, fibroblasts, brain, and other tissues. By knocking down different cathepsins, they too found that cathepsin L was necessary for progranulin processing, but so also was another lysosomal protease, cathepsin D. In agreement with Kukar’s data, inhibitors of lysosome acidification blocked progranulin processing. “I’m really happy that other people are seeing the same thing,” said Hu. “It makes me believe it’s real.”
These new studies should change how the field looks at progranulins, said Kukar. “The discovery that progranulin is constantly, rapidly trafficked to the lysosome and being turned over into granulins really surprised me,” he told Alzforum. Next, his group is working to test the role of individual granulins, to determine which if any might restore lysosome function in various models of granulin deficiency, and how they regulate lysosome homeostasis.
Malú Tansey, Emory University School of Medicine, Atlanta, called the work paradigm-shifting. “It goes against everything that we have assumed about progranulin in terms of function, and it makes sense. It will change the way we think about the disease, it will change the way we intervene, and I think that’s really exciting.” Tansey was not involved in the work.
Current therapeutic approaches to FTD-GRN focus on increasing progranulin levels (Nov 2014 conference news), but the new work suggests researchers should proceed with caution, especially if those strategies involve inhibiting lysosome processing or blocking uptake of exogenous progranulin into cells (Feb 2011 news). “If our hypothesis is true that you need progranulin to be internalized and processed into granulins to be functional, then compounds that boost progranulin by inhibiting that pathway aren’t going to cure the disease. That wasn't appreciated prior to our work,” Kukar said.
Kukar’s group is also working on new antibodies and quantitative assays for all granulins—the reagents used in the current study only detected three of the possible seven species.
The Kukar paper drew praise from several commenters for the extensive validation of antibodies and cell lines that preceded the actual experiments. “They deserve a huge amount of credit for doing the laborious, methodical experiments,” said Ward. “There are many interesting implications of this work, that the field is now well-positioned to address.”
Expanding the scope of progranulin’s actions in cells, the Sheng group reported a new role for the protein in autophagy, a key cell pathway for clearing large debris, including spent organelles and invading bacteria. In the study, progranulin knockout mice succumbed more readily to infection with listeria bacteria than their wild-type counterparts. The reason—a failure of autophagy, which normally clears the organisms. Because autophagosomes, the vesicles bearing cell debris, fuse with lysosomes to complete degradation of their contents, first author Michael Chang told Alzforum that the results fits with an overall negative effect of progranulin deficiency on these crucial degradation pathways. The lack of autophagy appeared to drive the accumulation of pathogenic forms of TDP-43 in cultured cortical neurons from knockout mice, and Chang showed that application of purified progranulin to the cells could normalize autophagy.
“We think all these papers emphasize the shift in thinking toward focusing on lysosome function. We think there is a unifying theme of many genes that cause FTD and ALS converging on lysosomal function and that will be a therapeutic area of increasing interest going forward,” Chang said.—Pat McCaffrey
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