Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 12;9(385):eaah5642.
doi: 10.1126/scitranslmed.aah5642.

Individuals With Progranulin Haploinsufficiency Exhibit Features of Neuronal Ceroid Lipofuscinosis

Affiliations
Free PMC article

Individuals With Progranulin Haploinsufficiency Exhibit Features of Neuronal Ceroid Lipofuscinosis

Michael E Ward et al. Sci Transl Med. .
Free PMC article

Abstract

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

Conflict of interest statement

Competing interests: L.G. has received honoraria from Sanford Burnham Prebys Medical Discovery Institute. W.W.S. has received consulting fees from Merck Inc. J.M.G. has received consulting fees from Genentech and MedImmune, medical legal consulting, and research support from Genentech, Quest Diagnostics, and MedDay. P.A. has received speaker honoraria, consultation fees, or travel support from Allergan, Biogen, Eisai, GlaxoSmithKline, Ipsen, Merz, Novartis, and Teva and research grants from Biogen, Ipsen, Merz, Novartis, and Roche. B.L.M. serves as the medical director for the John Douglas French Alzheimer’s Foundation; the scientific director for the Tau Consortium; the director/medical advisory board member of the Larry L. Hillblom Foundation; a scientific advisory board member for the National Institute for Health Research Cambridge Biomedical Research Centre and its subunit, the Biomedical Research Unit in Dementia (U.K.); and a board member for the American Brain Foundation. A.J.G. is a founder of Inception 5 Biosciences and is on the scientific advisory board of Bionure. All other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Retinal NCL-like storage material and lipofuscinosis in Grn knockout mice
(A) Representative EM images of retinal ganglion cell layer (GCL) neurons from 18-month-old wild-type (WT) and Grn knockout (Grn KO) mice. Note the characteristic fingerprint profile patterns of NCL-like storage material (Grn KO, inset). (B) Representative retinal cross section from 24-month-old Grn KO mice showing autofluorescent lipofuscin throughout the retina. Especially prominent lipofuscinosis occurs in the GCL, inner nuclear layer (INL), deep layers of the inner plexiform layer (IPL), outer plexiform layer (OPL), and outer nuclear layer (ONL). Additionally, subretinal drusen-like autofluorescent aggregates occurred in Grn KO mice (white arrow). DAPI, 4′,6-diamidino-2-phenylindole; IS, inner segment; OS, outer segment. (C) Representative lipofuscin deposits (red puncta) in retinal whole mounts from aging WT and Grn KO mice. (D) Quantification of lipofuscin deposits from (C) n = 4 to 26 mice per age per genotype. ***P < 0.001, one-way analysis of variance (ANOVA) with Tukey’s post hoc test. (E) Representative images of retinal lipofuscin in Grn KO mice imaged in vivo using cSLO. Green lines overlaid on cSLO images indicate the location of cross-sectional OCT scans. Occasional subretinal drusen-like deposits were observed in Grn KO mice (insets 1 and 2, red arrow), but most autofluorescent puncta were below the limit of detection of OCT cross-sectional scans through the same area (insets 3 and 4). (F) Quantification of in vivo imaging of retinal lipofuscin from (E). n = 11 to 12 eyes from six mice per genotype. ***P < 0.001, mixed-effect multivariate linear regression model.
Fig. 2
Fig. 2. Autofluorescent NCL-like storage materialin theCNS of humans with heterozygous GRN mutations
(A to C) In vivo imaging of retinal lipofuscinosis in humans with GRN haploinsufficiency. Representative images of cSLO autofluorescent retinal imaging of one control subject (Ctrl) and two GRN mutation carriers (GRN mt) are shown. OCT cross-sectional scans (green dashed lines) are shown below the autofluorescent images. Lipofuscin deposits (insets) occurred more frequently in GRN mutation carriers than in control subjects. Most lipofuscin deposits did not correlate with detectable abnormalities in OCT cross-sectional scans (B), although some correlated with subretinal drusen-like material (C, red arrows). Scale bars, 1 mm. (D to F) Blinded quantification of retinal autofluorescence images obtained by cSLO. Data are grouped by all GRN mutation subjects versus all controls (n = 11 and 22), presymptomatic GRN mutation subjects versus matched controls (n = 8 and 16), and symptomatic GRN mutation subjects versus matched controls (n = 3 and 6). (D) Heterozygous GRN mutation carriers were more likely than controls to have retinal lipofuscin deposits; P = 0.027, Fisher’s exact test. Asymptomatic and symptomatic GRN mutation carriers also were more frequently found to have retinal lipofuscin deposits, although the difference was not significant. P = 0.18and0.46, respectively, Fisher’s exact test. The number of affected subjects is shown inside the bars. *P < 0.05. (E) Quantification of the total number of retinal lipofuscin deposits per subject. Heterozygous GRN mutation carriers had more lipofuscin deposits than did controls, including sub-groups of asymptomatic and symptomatic GRN mutation carriers. *P <0.05, ***P < 0.001, Mann-Whitney U test. (F) Quantification of the total area of retinal lipofuscin deposits per subject. Heterozygous GRN mutation carriers had a greater total area of lipofuscin deposits than did controls, including a subgroup of asymptomatic GRN mutation carriers. **P < 0.01, Mann-Whitney U test. N.S., not significant. (G) Increased accumulation of electron-dense storage material in postmortem cortical tissue from heterozygous GRN mutation carriers. Shown are representative electron micrographs of formalin-fixed frontal cortex from control and heterozygous GRN mutation carriers. Note the presence of granular osmiophilic deposit–like storage material in neurons in cortical tissue from GRN mutation carriers (insets, yellow arrow), which was rarely observed in control cortical neurons. (H) Quantification of electron-dense storage material seen in (G). The percentage of neurons that contained such storage material and the number of deposits per neuron are shown. n = 4 to 26 neurons from 15 GRN mutation carriers, 16 control subjects, and 6 AD subjects. Percentage of neurons: ***P < 0.001, one-way ANOVA with Bonferroni’s multiple comparison test; number of deposits per cell: **P < 0.001, mixed model linear regression. (I) Increased autofluorescent lipofuscin aggregates in postmortem cortical tissue from heterozygous GRN mutation carriers. n = 6 to 30 fields of view from nine GRN mutation carriers and nine controls; **P = 0.009, mixed-model linear regression. (J) Representative bright-field images of frontal cortex showing an increase in Sudan black–stained lipofuscin in postmortem cortical tissue from a heterozygous GRN mutation carrier versus control. (K) Quantification of Sudan black staining seen in (J). The number of lipofuscin deposits per field of view is shown. n = 10 GRN mutation carriers and 14 control subjects; ****P < 0.0001, Student’s t test. Means ± SEM are shown in (E), (F), (H), (I), and (K).
Fig. 3
Fig. 3
GRN haploinsufficiency results in NCL-like storage material in lymphoblasts. (A) NCL-like storage material accumulates in lymphoblasts from a heterozygous GRN mutation carrier who clinically presented with NCL symptoms. Representative images of lymphoblasts from a healthy non–mutation-carrying control subject, an NCL patient (homozygous CLN3 mutation carrier), and a heterozygous GRN mutation carrier are shown. Note the similar ultrastructural patterns of the storage material observed in the CLN3 mutation carrier and the GRN mutation carrier, consisting of prominent vacuolated structures containing storage material with a fingerprint profile pattern (insets). (B) Quantification of NCL-like storage material in lymphoblasts from heterozygous GRN mutation carriers without clinical evidence of NCL versus noncarrier sibling controls. Five paired sets of lymphoblasts from control and heterozygous GRN mutation siblings were imaged with EM, and the number of vacuoles with storage material per cell and cross-sectional area occupied by storage material containing vacuoles was quantified in a blinded manner. n = 19 to 38 cells per subject from five pairs of GRN mutation carrier and control subjects; ***P < 0.001 via mixed-effect multivariate linear regression model. (C to G) Restoring normal PGRN expression in lymphoblasts from heterozygous GRN mutation carriers rescued the NCL-like storage material phenotype. (C) Western blot of intracellular PGRN and tubulin showing reduced expression of PGRN in heterozygous GRNExon 8 (IVS7-1g) mutation carrier lymphoblasts, which was rescued by treatment with a PGRN-expressing retrovirus. Control (empty vector) retrovirus did not alter PGRN expression. GRNExon 8 (IVS7-1g) mutation carrier lymphoblasts transduced with the PGRN-retrovirus that expressed PGRN at two different levels (endogenous, +; high, ++) were used for EM analysis. (D) Quantification of PGRN expression (normalized to tubulin). n = 3 wells per group; *P < 0.05, **P < 0.01, one-way ANOVA with Sidak’s multiple comparison test. (E) Representative electron micrographs of heterozygous GRNExon 8 (IVS7-1g) mutation carrier lymphoblasts transduced with control (empty vector) or huPGRN retrovirus expressing PGRN at an endogenous level. Lymphoblasts transduced with PGRN-expressing retrovirus had less storage material than did those that were treated with empty vector (insets). (F and G) Quantification of the number of vacuoles with storage material per lymphoblast (F) or total cross-sectional area of storage material per lymphoblast (G) in lymphoblasts transduced with control or PGRN-expressing retrovirus. n = 45 to 65 cells per group, *P < 0.05, **P < 0.01, one-way ANOVA with Sidak’s multiple comparison test. Means ± SEM are shown (B, D, F, and G).
Fig. 4
Fig. 4. Impaired lysosomal protease activity in primary cells from heterozygous GRN mutation carriers
(A) Representative images of fluorescence after staining with BMV109 (red), a cell-permeable probe for active cysteine cathepsin proteases, in HDFs from heterozygous GRN mutation carrier and control siblings (blue, DAPI nuclear stain). (B) Quantification of BMV109 fluorescence in heterozygous GRN mutation carrier and control HDFs. n = 6 wells imaged per HDF line from three pairs of GRN mutation carrier and matched sibling controls; ****P < 0.0001 via mixed-effect multivariate linear regression model. AU, arbitrary units. (C) Cathepsin D enzyme activity in HDF lysates from heterozygous GRN mutation carrier and control sibling pairs. n = 10 assays per HDF line from three pairs of GRN mutation carrier and matched sibling controls; ****P < 0.0001 via mixed-effect multivariate linear regression. Mean enzyme activity, normalized to matched sibling control lines, ±SEM is shown (B and C).

Similar articles

See all similar articles

Cited by 32 articles

See all "Cited by" articles

MeSH terms

Substances

Feedback