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. 2015 Jun 24;35(25):9315-28.
doi: 10.1523/JNEUROSCI.4808-14.2015.

The Progranulin Cleavage Products, Granulins, Exacerbate TDP-43 Toxicity and Increase TDP-43 Levels

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Free PMC article

The Progranulin Cleavage Products, Granulins, Exacerbate TDP-43 Toxicity and Increase TDP-43 Levels

Dominique A Salazar et al. J Neurosci. .
Free PMC article

Abstract

Mutations in the human progranulin gene resulting in protein haploinsufficiency cause frontotemporal lobar degeneration with TDP-43 inclusions. Although progress has been made in understanding the normal functions of progranulin and TDP-43, the molecular interactions between these proteins remain unclear. Progranulin is proteolytically processed into granulins, but the role of granulins in the pathogenesis of neurodegenerative disease is unknown. We used a Caenorhabditis elegans model of neuronal TDP-43 proteinopathy to specifically interrogate the contribution of granulins to the neurodegenerative process. Complete loss of the progranulin gene did not worsen TDP-43 toxicity, whereas progranulin heterozygosity did. Interestingly, expression of individual granulins alone had little effect on behavior. In contrast, when granulins were coexpressed with TDP-43, they exacerbated its toxicity in a variety of behaviors including motor coordination. These same granulins increased TDP-43 levels via a post-translational mechanism. We further found that in human neurodegenerative disease subjects, granulin fragments accumulated specifically in diseased regions of brain. To our knowledge, this is the first demonstration of a toxic role for granulin fragments in a neurodegenerative disease model. These studies suggest that presence of cleaved granulins, rather than or in addition to loss of full-length progranulin, may contribute to disease in TDP-43 proteinopathies.

Keywords: C. elegans; TDP-43; frontotemporal lobar degeneration; granulin; neurodegenerative disease; progranulin.

Figures

Figure 1.
Figure 1.
Progranulin gene dosage differentially affects behavior in TDP-43 animals. A, TDP-43 expression impairs motor function as measure by thrashes per 30 s. Two independent lines of animals expressing TDP-43 were tested. These lines exhibited similar results in all assays tested; therefore, data from only one is shown in subsequent figures. Data represents average of 3 trials (n = 12 per trial). B, C, TDP-43 expression did not affect pharyngeal contractions (B) or rate of defecation (C; n = 15 per trial). D, To measure development, the larval stage of each animal was scored at 48 h after the 100-cell stage. Data are graphed as percentage developing to each larval stage after 48 h (n = 50). E, Lifespan is unaffected by TDP-43 expression (Mantel–Cox test). Data shown corresponds to trial 1 of 3 in Table 2. F, pgrn-1(+/−); TDP-43 animals exhibit impaired motor coordination compared with the wild-type N2 strain (n ≥ 11). For all panels: ns, not significant; wt, wild-type; mut, mutant; het, heterozygote; *p < 0.05, **p < 0.01 by one-way ANOVA. Error bars represent SD.
Figure 2.
Figure 2.
Coexpression of TDP-43 with granulin 2 or 3 synergistically delays larval development. A, Models of Homo sapiens versus C. elegans progranulin protein and FLAG-tagged granulin constructs (TM domain, transmembrane domain). B, Anti-FLAG Western blot (top) and quantification (bottom) demonstrates granulin expression in indicated strains. No significant differences are found (n = 3 biological replicates; one-way ANOVA; error bars represent SD). C, Gran1::GFP, gran2::GFP and gran3::GFP are all localized in a subset of vesicles in intestinal cells. Representative images from each line are shown. Scale bar, 10 μm. Arrowheads indicate intestinal nuclei. D, All three granulins were efficiently secreted from their cells of origin and taken up by coelomocytes, which do not produce progranulin (Kao et al., 2011). Representative images of granulin::GFP in coelomocytes is shown. Scale bar, 10 μm. Arrows indicate posterior coelomocytes.
Figure 3.
Figure 3.
TDP-43 and granulins 2 and 3 synergistically delay development, impair growth, and increase bacterial packing. A, Granulin 2 and 3 delay development when coexpressed with TDP-43 (n = 50 per strain). B, Representative micrographs from each strain at L4 stage show that animals reach similar size. C–E, Measurement of animal size at Day 5 of adulthood. Representative images of a wild-type (C) and TDP-43;gran3 (D) worms. Animals <2 SD below the wild-type mean are score scored as small size. Results are shown in E (n = 12). F–H, Representative images of a normal pharynx from a wild-type animal (F) and abnormal bacterial packing in a TDP-43;gran3 animal (G) are shown. Scores for bacterial packing in each strain tested are shown in H (n = 12).
Figure 4.
Figure 4.
Animals expressing TDP-43 with granulin 2 or 3 exhibit circuit-specific neuronal dysfunction. A, Granulin 2 and 3 impair motor coordination when coexpressed with TDP-43. Data are averages from 3 separate trials (n = 12 per trial). TDP-43 with coinjection marker alone was tested and had no effect on thrashing compared with TDP-43 alone (data not shown). B, Coexpression of full-length progranulin (Ppgrrn-1::pgrn-1::rfp) with TDP-43 in a pgrn-1(-) background does not alter motor function (n = 12 per trial). C, Endogenous progranulin does not ameliorate motor defects caused by coexpression of granulins 2 and 3 with TDP-43 (n = 12 per trial). D, Granulins did not impair defecation in TDP-43 animals (n = 15 per trial). E, Granulins did not impair pharyngeal function in TDP-43 animals (n = 15 per trial). F, Pharyngal pumping was measured at Day 5 of adulthood as previously described. No significant differences were seen between strains. Data shown represents one trial (n = 6 per trial). For all panels: ns, not significant; wt or w, wild-type; mut or m, mutant; ***p < 0.0001 by one-way ANOVA. Error bars represent SD. Unless otherwise indicated, data are from Day 1 adult animals.
Figure 5.
Figure 5.
Coexpression of TDP-43 and specific granulins reduces lifespan and alters reproductive efficiency. A, Strains expressing both TDP-43 and granulin 3 had a median lifespan of 8 d, compared with 16 d for wild-type (n = 90). Shown is trial 1 of two independent trials (Table 5). B, Percentage of animals with internally hatched progeny for each strain.
Figure 6.
Figure 6.
Expression of TDP-43 with granulins 2 and 3 results in a constitutive egg-laying (Egl-C) phenotype. A, Animals were treated with 5 mg/ml serotonin hydrochloride (5HT) for 1 h and then eggs laid were quantified (n = 8 per strain). B, Animals were treated with M9 for 1 h and then eggs laid were quantified. Granulin; TDP-43 animals exhibited constitutive egg laying (n = 8 per strain). C, The percentage of early-stage eggs laid by Day 1 adults from each strain. Granulin; TDP-43 animals exhibited premature egg laying (data shown are the average of two trials, n ≥ 100 per trial). For all panels: ns, not significant; wt, wild-type; mut, mutant; *p < 0.05, **p < 0.01 by one-way ANOVA. Error bars represent SEM.
Figure 7.
Figure 7.
Granulins 2 and 3 promote TDP-43 accumulation. A, Intact DA/DB motor neurons (cell bodies and axons) were quantified for pgrn-1(-) and TDP-43;gran 3 animals expressing the Punc-129::GFP neuronal marker. No significant differences were found (n = 10). B, VC neurons (cell bodies and axons) were counted for pgrn-1(-) and TDP-43;gran 3 animals expressing the Plin-11::GFP neuronal marker. No significant differences were found (n = 10). C, Images of TDP-43::GFP nuclear localization in Day 1 adult worms. Arrows indicate neuronal nuclei. TDP-43 was observed only in nuclei and not in neuronal cell bodies or extensions. D, Western blot against total TDP-43 and phospho-TDP-43 (S409/S410) for indicated strains. E, Quantitation of total TDP-43 from Western blots, normalized to actin loading control. Data shown represents the average of three independent trials (unpaired Mann–Whitney test, single tail, *p < 0.05; error bars represent SEM). F, TDP-43 mRNA levels were determined by qPCR using two different primer sets for TDP-43. Shown is the fold-change in expression compared with the pgrn-1;TDP-43 strain. No significant differences were observed (n = 3 independent biological replicates, one-way ANOVA, error bars represent SD). G, Granulin does not affect deg-1(d)-induced touch cell necrosis. Necrotic PVC touch receptor neurons were quantified in strains through development and the percentage of animals demonstrating necrotic cells determined (n ≥ 120, error bars indicate SEM). H, Granulin does not further impair motor control in tau transgenic strains (n = 12; ns, not significant, one-way ANOVA; error bars represent SD).
Figure 8.
Figure 8.
A granulin cleavage product is over-represented in diseased brain regions from AD and FTLD patients. A, Anti-granulin E and anti-actin Western blots of postmortem brain tissue from control subjects or patients with pathological diagnosis of AD or FTLD-TDP-A. Tissue was sampled from diseased regions with high gliosis (middle frontal gyrus, MFG) and nondiseased areas with low gliosis (CC) in AD and FTLD subjects and the same areas in control individuals. A ∼33 kDa band is marked by arrowheads. See Table 6 for clinical information. B, Quantification of the ∼33 kDa fragment from control or neurodegenerative disease subjects normalized to actin. Shown is the fold-change in signal intensity in CC compared MFG (*p = 0.023, Student's t test). C, Anti-granulin E Western blot of C. elegans strains expressing human progranulin tagged with mCherry. Arrowheads indicate specific bands, open arrowhead indicates a granulin E cleavage fragment.

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