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Case Reports
. 2020 Mar 1;143(3):783-799.
doi: 10.1093/brain/awaa039.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis

Carol Dobson-Stone  1   2   3 , Marianne Hallupp  1   2 , Hamideh Shahheydari  4 , Audrey M G Ragagnin  4 , Zac Chatterton  1   5   6 , Francine Carew-Jones  2   3 , Claire E Shepherd  2   3 , Holly Stefen  7 , Esmeralda Paric  7 , Thomas Fath  7 , Elizabeth M Thompson  8   9 , Peter Blumbergs  10 , Cathy L Short  11 , Colin D Field  12 , Peter K Panegyres  13 , Jane Hecker  14 , Garth Nicholson  15   16   17 , Alex D Shaw  1   2   3 , Janice M Fullerton  2   3 , Agnes A Luty  2   3 , Peter R Schofield  2   3 , William S Brooks  2   18 , Neil Rajan  19 , Mark F Bennett  20   21   22 , Melanie Bahlo  20   22 , John E Landers  23 , Olivier Piguet  24   25 , John R Hodges  1   25 , Glenda M Halliday  1   2   3 , Simon D Topp  26 , Bradley N Smith  26 , Christopher E Shaw  26 , Emily McCann  4 , Jennifer A Fifita  4 , Kelly L Williams  4 , Julie D Atkin  4   27 , Ian P Blair  4 , John B Kwok  1   2   3
Affiliations
Case Reports

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis

Carol Dobson-Stone et al. Brain. .

Abstract

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Keywords: CYLD; autophagy; deubiquitinase; genome-wide linkage analysis; whole-exome sequencing.

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Figures

Figure 1
Figure 1
Family Aus-12 has a shared disease haplotype at chromosome 16p12.3–16q12.2. Black symbols show individuals with presenile dementia: clinically diagnosed as Alzheimer’s disease (Individual II:2), FTD (Individuals IV:5, IV:7 and IV:23), or unspecified dementia (Individuals I:2, III:1, III:7, III:9, III:12, III:17, IV:4 and IV:6); grey symbols show individuals with ALS. A diagonal line indicates deceased subjects. Individuals with DNA available are indicated with an asterisk. Inferred haplotypes are in parentheses. Allele data for 12 microsatellite markers and single nucleotide variants in the region are shown, including g.50825515A>G (= CYLD p.M719V).
Figure 2
Figure 2
CYLDM719V affects CYLD immunoreactivity in brain tissue. Photomicrographs demonstrating CYLD immunoreactivity in the frontal cortex and hippocampus. (A) Parahippocampal white matter showing CYLD peroxidase labelling of blood vessels, which was seen in all cases. (B) CYLD staining was seen in balloon neurons in all cases. Image shows representative image taken from Sydney Brain Bank Case 2. (C and D) CYLD-positive glia were common in Family Aus-12 (D) but not sporadic cases (C). Images were taken in the frontal white matter of Sydney Brain Bank Case 1 and Individual IV:7, respectively. (E) Diffuse, neuronal cytoplasmic CYLD staining of a neuron in the hippocampus of Individual IV:5. (F) CYLD-positive nuclear staining of a pyknotic neuron in the deep layers of the frontal cortex of Individual IV:7. (G and H) Double labelling immunofluorescence in the dentate gyrus of Individuals IV:5 (G) and IV:7 (H). TDP-43 (green, arrowheads in G) and Tau (green, H) did not co-localize with CYLD (red, G and H) in any case. Scale bar = 100 μm in A, 20 μm in B and E–H, 40 μm in C and D.
Figure 3
Figure 3
Expression of CYLDM719V alters neuronal TDP-43 localization. (A) Representative fluorescence images of mouse cortical neurons at 7 days in vitro, overexpressing GFP or GFP-tagged CYLDwt, CYLDD681G or CYLDM719V (green). TDP-43 was detected by immunofluorescent staining (red) and nuclei were visualized with DAPI (blue). Arrowheads indicate cytoplasmic TDP-43 staining in cells transfected with CYLDwt and CYLDM719V. (B) Quantification of the percentage of transfected cells with observable cytoplasmic TDP-43 staining, showing a significantly higher proportion of cytoplasmic TDP-43-positive cells in the CYLDM719V condition relative to CYLDwt. Horizontal bars indicate mean values from three independent experiments. Scale bars = 10 μm. **P < 0.005, ****P < 0.00005.
Figure 4
Figure 4
Expression of CYLDM719V alters axonal morphology. (A1–D1) Representative fluorescence images of mouse hippocampal neurons at 6 days in vitro, overexpressing GFP (A1), CYLDwt-GFP (B1), CYLDD681G-GFP (C1) or CYLDM719V-GFP (D1). (A2–D2) Inverted black and white images of GFP fluorescence of the transfected neurons shown in A1–D1, with pseudo-coloured axon labelling for clearer visualization of the axonal compartment. (E–H) Quantification of axonal morphology. Box plots represent median, interquartile range and minimum-maximum (n = 20 each group). *P < 0.05, **P < 0.005. Scale bars = 200 μm.
Figure 5
Figure 5
CYLDM719V shows increased K-63 deubiquitinase activity and inhibition of NF-κB. K63-linked ubiquitin tetramers (Ub4) were incubated with wild-type (wt) or M719V mutant CYLD-mycFLAG and digestion was observed. (A) Representative immunoblot showing appearance of bands corresponding to ubiquitin trimers (Ub3) and dimers (Ub2) after 15, 30 and 45 min of CYLD treatment. An uncropped image of this blot is available in Supplementary Fig. 4. (B) Quantification of ubiquitin cleavage expressed as log10-transformed Ub4/Ub2 after treatment with CYLDwt (open circles) or CYLDM719V (filled squares) (mean ± SEM from 10 experiments). (C) Comparison of rate of K63-ubiquitin cleavage shows significantly increased rate of cleavage for CYLDM719V relative to CYLDwt. Box plots represent median, interquartile range and minimum-maximum from n = 10 independent experiments. (D) Quantification of NF-κB activity. HEK293 cells were transfected with NF-κB-responsive luciferase construct pGL4.32 plus empty vector or human (h) CYLDwt, CYLDD681G or CYLDM719V constructs or mouse (m) equivalents Cyldwt, CyldD680G or CyldM718V, and NF-κB expression was induced by tumour necrosis factor α treatment. Horizontal bars indicate mean values from five independent experiments. Values are normalized to human CYLDwt or mouse Cyldwt, and show significant increases for cylindromatosis mutant CYLDD681G and mouse equivalent CyldD680G and decreases for Family Aus-12 mutant CYLDM719V and mouse equivalent CyldM718V. **P < 0.005, ***P < 0.0005.
Figure 6
Figure 6
CYLDM719V impairs fusion of autophagosomes with lysosomes. (A) Images of HEK293 cells cotransfected with tandem mCherry-GFP-tagged autophagy marker LC3 plus empty vector or mycFLAG-tagged CYLDwt, CYLDD681G or CYLDM719V constructs. Cells were serum starved for 6 h to induce autophagy. Fusion of the autophagosome with the lysosome to produce autolysosomes quenches GFP but not mCherry fluorescence. In the merged image, autophagosomes appear as yellow vesicles, while autolysosomes appear as red vesicles. (B) Quantification of the percentage of autophagosomes (mCherry- and GFP-positive vesicles, left) and autolysosomes (mCherry-positive only vesicles, right) relative to total vesicle number, showing a significant decrease in proportion of autolysosomes in CYLDM719V cells, but not CYLDD681G cells relative to CYLDwt. Box plots represent median, interquartile range and minimum-maximum (n = 88 each group). Scale bar = 10 μm. ***P < 0.0001.
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