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. 2010 Mar;112(5):1305-15.
doi: 10.1111/j.1471-4159.2009.06546.x. Epub 2009 Dec 17.

Pathogenic cysteine mutations affect progranulin function and production of mature granulins

Jun Wang  1 , Philip Van Damme, Carlos Cruchaga, Michael A Gitcho, Jose Manuel Vidal, Manuel Seijo-Martínez, Lei Wang, Jane Y Wu, Wim Robberecht, Alison Goate
Affiliations

Pathogenic cysteine mutations affect progranulin function and production of mature granulins

Jun Wang et al. J Neurochem. 2010 Mar.

Abstract

Frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) can be caused by mutations in the progranulin gene (GRN). Progranulin (PGRN) is a cysteine-rich growth factor, which is proteolytically cleaved by elastase to produce several granulins (GRNs). All FTLD-U mutations in GRN characterized to date result in reduced secreted PGRN protein. We recently reported a Spanish family with progressive non-fluent aphasia and dementia in which a novel C521Y mutation segregates with disease. A second cysteine mutation (C139R) has also been reported to be disease specific. Allele-specific mRNA expression assays in brain reveal that the C521Y mutant allele is expressed at similar levels to the wild-type allele. Furthermore, plasma PGRN levels in C521Y carriers are comparable with non-carrier family relatives, suggesting that the mutation does not affect PGRN protein expression and secretion in vivo. Despite normal PGRN levels C521Y and C139R mutant GRNs show reduced neurite growth-stimulating activity in vitro. Further study revealed that these mutations also cause impaired cleavage of PGRN by elastase. Our data suggest that these mutations affect the function of full-length PGRN as well as elastase cleavage of PGRN into GRNs, leading to neurodegeneration.

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Figures

Figure 1
Figure 1. Plasma PGRN levels in GRN mutation carriers and non-carriers in PNFA (A) and HDDD2 (B) families
PGRN levels in plasma samples from PNFA family (5 C521Y mutation carriers and 2 non-carriers) and HDDD2 family (4 A9D mutation carriers and 4 non-carries) were measured using a human Progranulin ELISA kit, according to the Manufacturer’s instructions. The bar diagram represents mean values ± SEM. Asterisks indicate a significant difference between A9D-carriers and HDDD2 family controls, determined by Student’s t-test (*** p < 0.001). There is no difference between PNFA family non-carriers and C521Y-carriers (p=0.847).
Figure 2
Figure 2. The C521Y and C139R mutations do not affect PGRN expression and secretion in a transfection system
A. HEK cells were transfected with GFP, WT-PGRN or mutant PGRNs as indicated. Cell lysates (5 μg) and conditioned media (5 μl) were separated on a 4-12% Bis-Tris gel under reducing conditions. The membrane was western blotted with anti-PGRNC-term (upper panel). The same membrane was stripped and reblotted with an anti-actin antibody (lower panel). B, C. Semi-quantitative densitometric analysis. B. The densities of the PGRN bands (lysates) were quantified using Quantity One Software (Bio-rad) and normalized to actin levels. %WT was plotted. C. Percentages of normalized PGRN in media over total (media + lysates) were plotted.
Figure 3
Figure 3. The cysteine mutation affects protein mobility under non-reducing conditions, but not reducing conditions
Media from HEK cells transfected with WT, C521Y or C139R were collected and subjected to PNGase F treatment. Samples were mixed with non-reducing (N) or reducing (R) sample buffers, separated on a 4-12% Bis-Tris gel and western blotted with anti-PGRNC-term.
Figure 4
Figure 4. The C139R and C521Y mutant PGRN do not stimulate neurite outgrowth
A. 10 μl conditioned media from HEK cells stably expressing vector control (CTR), C139R, C521Y and wtPGRN was resolved in 7.5% SDS-Tris gel and western blotted with a monoclonal anti-GFP antibody. B. Primary cultures of mouse cortical neurons were grown in the presence of 1 ml conditioned media from HEK cells expressing vector control (CTR), wtPGRN or PGRN mutants C139R and C521Y. Axonal outgrowth assays were performed as described in Materials and Methods. Shown are representative neurons visualized with neuronal βIII tubulin (TUJ1, Red, 1), nuclei (Nu, Blue, 2) and Merge (3). C. Quantification of axon outgrowth. The average length of the longest neurite was quantified using ImageJ software from 200 randomly selected neurons in each group. The data represent three independent experiments. The bar diagram represents mean values ± SEM. Asterisks indicate a significant difference from the wtPGRN, as determined by Student’s t-test (*** p < 0.001).
Figure 5
Figure 5. The cysteine mutations affect PGRN cleavage by elastase
A. Serum-free media collected from HEK cells expressing WT, C521Y or C139R PGRN were treated with saline (S) for 90 min or elastase for various times as indicated and resolved on a 4-20% Tris-HCl gel under reducing conditions. The membrane was blotted with anti-PGRNC-term. Arrows indicate the 35 kD, 19 kD and 10 kD C-terminal fragments detected by anti-PGRNC-term. B. Same as A, except that the samples were run under non-reducing conditions and immunoblotted with anti-PGRNFL. Arrows indicate the 45 kD, 35 kD and 25 kD fragments detected by this antibody. C. 1.25 μg of recombinant GST tagged WT or C521Y GRN E peptides (GST-GRN E and GST-C521Y) were run on a 4-20% Tris-HCl gel under reducing conditions and western blotted with anti-PGRNC-term.
Figure 6
Figure 6. The C521Y mutant peptide promotes neuronal survival and neurite outgrowth
A. Effect of GST-GRN E and GST-C521Y peptides on survival of cortical neurons on day 2 in culture measured by a calcein assay, normalized to untreated controls (control, n = 10, * different from control p < 0.03). B. Effect of GST-GRN E and GST-C521Y peptides on survival of motor neurons on day 2 in culture (n = 6, * different from control p < 0.005). C. Effect of GST-GRN E and GST-C521Y peptides on soma size in cortical and motor neurons (n = 58-106, p > 0.3). D. Effect of GST-GRN E and GST-C521Y peptides on neurite length of longest neurite per cell (n = 58-106, * different from control p ≤ 0.001). Error bars show mean ± SEM. There is no significant difference between GST-GRN E and GST-C521Y.
Figure 7
Figure 7. A model of PGRN processing by elastase
Only fragments detected in this study are shown. Full-length PGRN protein is initially cleaved by elastase at linker AC (solid arrow) to yield a 45 kD N-terminal intermediate fragment (encompassing GRNs P-G-F-B-A) and a 35 kD C-terminal intermediate fragment (encompassing GRNs C-D-E). The 35 kD intermediate fragment is further cleaved at linkers CD and DE (solid arrows) to yield a 19 kD fragment (GRNs D-E) and a 10 kD fragment (GRN E) that are detected with anti-PGRNC-term. The 45 kD intermediate fragment is further cleaved possibly at linker FB (dashed arrow), to produce a 25 kD fragment detected by anti-PGRNFL. A dashed arrow is used because the exact site of this cleavage is not confirmed in this study.
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