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. 2016 Nov 3;99(5):1117-1129.
doi: 10.1016/j.ajhg.2016.09.010. Epub 2016 Oct 20.

Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant

Nataliya Di Donato  1 , Ying Y Jean  2 , A Murat Maga  3 , Briana D Krewson  4 , Alison B Shupp  4 , Maria I Avrutsky  2 , Achira Roy  5 , Sarah Collins  5 , Carissa Olds  5 , Rebecca A Willert  4 , Agnieszka M Czaja  4 , Rachel Johnson  4 , Jessi A Stover  4 , Steven Gottlieb  6 , Deborah Bartholdi  7 , Anita Rauch  8 , Amy Goldstein  9 , Victoria Boyd-Kyle  4 , Kimberly A Aldinger  5 , Ghayda M Mirzaa  10 , Anke Nissen  11 , Karlla W Brigatti  12 , Erik G Puffenberger  12 , Kathleen J Millen  5 , Kevin A Strauss  13 , William B Dobyns  14 , Carol M Troy  15 , Robert N Jinks  16
Affiliations

Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant

Nataliya Di Donato et al. Am J Hum Genet. .

Abstract

Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thickening and reduced gyration. Here we describe a "thin" lissencephaly (TLIS) variant characterized by megalencephaly, frontal predominant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-β-induced dendritic spine collapse and neuronal apoptosis, suggesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD's ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mechanism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability.

Keywords: MCD; apoptosis; epilepsy; intellectual disability; malformation of cortical development; mouse model; neurodevelopmental disorder; pachygyria.

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Figures

Figure 1
Figure 1
Brain Imaging of TLIS-Affected Individuals with CRADD Mutations (A–D) MRI in subject LR04-101a1 at 5 years shows normal brainstem and cerebellum (A, B), frontal predominant symmetric pachygyria with reduced number of gyri, shallow sulci, and mildly thickened cortex (double arrowheads in B, C, and D with arrowheads located only in the left hemisphere). (E–H) MRI in subject LR05-279a2 at 7 years demonstrates the same pattern as in the top row (double arrowheads in F, G, and H). The enlargement in the inferior cerebellum is a mild Chiari malformation type I (asterisk in E). (I–L) Age-matched normal control images. The cortex in TLIS-affected individuals is mildly thick, measuring 5–7 mm in the frontal lobes (see C and G) compared to normal cortex, measuring 3–4 mm (inset in K). MRI image section planes: A, B, E, F, I, J, sagittal T1-weighted images; C, G, K, axial T1-weighted images; D, H, L, coronal T2-weighted images.
Figure 2
Figure 2
TLIS CRADD Variants Do Not Disrupt Binding with PIDD or Caspase-2 (A) Functional domain structure of CRADD showing TLIS substitutions clustered in the C-terminal death domain (DD, amino acids 116–188). CARD, N-terminal caspase-recruitment domain (amino acids 1–91). CRADD substitutions p.Gly128Arg, p.Arg170Cys, and p.Arg170His are homozygous in individuals with TLIS. The p.Phe164Cys encoding CRADD allele (c.491T>G) is in trans with a 3.07 Mb deletion of chromosome 12q22 (denoted by the two red Xs) in one TLIS-affected subject (see Table 1). (B) Immunoblot (IB) of recombinant FLAG-CRADD TLIS variants overexpressed in HEK293T cells (n = 6). NTC, non-transfected control. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1 [MIM: 603453]; 78 kDa) immunoblotting was used as a loading control. (C) Top: western blot of CRADD (23 kDa) from dermal fibroblasts of a TLIS-affected subject homozygous for p.Gly128Arg CRADD (p.Gly128Arg/p.Gly128Arg) versus heterozygous parent (WT/p.Gly128Arg) and normal human dermal fibroblasts (WT/WT) (n = 4). Anti-β-actin (42 kDa) immunoblotting was used as a loading control. Bottom: Reverse transcriptase-PCR of the full-length human CRADD coding sequence (GenBank: NM_003805.3; 658 bp) from CRADD p.Gly128Arg (c.382G>C) TLIS-affected subject dermal fibroblasts (p.Gly128Arg/p.Gly128Arg) versus heterozygous parent (WT/p.Gly128Arg) and normal fibroblasts (WT/WT) demonstrating stability of the mutant transcript. Note: the WT/WT lane of the RT-PCR gel was cropped from the same gel used for the subject and heterozygous parent samples. Cropping is denoted by the white bar separating lanes 2 and 3. (D) FLAG-CRADD WT and TLIS variants were co-overexpressed with V5-PIDD-DD in HEK293T cells (as indicated) for 40 hr after which FLAG-CRADD was immunoprecipitated (IP) from whole cell lysates (input) with anti-FLAG M2 affinity resin. Co-precipitated complexes were first immunoblotted (IB) with anti-V5 antibody (PIDD-DD). Blots were then stripped and reprobed with anti-FLAG antibody (CRADD). CRADD WT and each of the TLIS CRADD variants except for p.Gly128Arg CRADD co-precipitated PIDD-DD (n = 3). (E and F) Immunoprecipitation (IP) of caspase-2 from PC12 cells 5 hr after the cells were transduced with 27 nM Pen1-CRADD proteins as indicated (n = 2–3). Caspase-2 co-precipitated Pen1-CRADD WT and TLIS variants in similar abundances when corrected for Pen1-CRADD abundance in whole cell lysates (input) as shown in (F). Blots were stripped and reprobed with anti-caspase-2. Error bars represent SEM. NS, no significant difference; control, non-transduced cells. ERK immunoblotting was used as an input lysate loading control. CRADD p.Gly128Arg abundance is always lower in transduced PC12 cells, in agreement with our previous findings.
Figure 3
Figure 3
TLIS-CRADD Variants Fail to Activate Caspase-2-Initiated Apoptosis (A and B) Pen1-CRADD WT (27 nM) drives apoptosis 1 day after transduction in PC12 cells (A) and in primary sympathetic neurons from Cradd−/− mice (B) whereas Pen1-TLIS-CRADD variants (27 nM) do not (n = 4 for each cell type). Control indicates non-transduced cells. (C) TLIS-CRADD variants fail to activate caspase-2. PC12 cells were treated with 50 μM bVAD-fmk for 1 hr followed by transduction with 27 nM Pen1-CRADD as indicated for 2 hr. Caspase-bVAD-fmk complexes in whole-cell lysates were precipitated with streptavidin and immunoblotted for caspase-2. Precipitated caspase-2 band optical density was used as a measure of relative caspase-2 activity, (n = 3). Error bars represent SEM. Control indicates non-transduced cells.
Figure 4
Figure 4
Megalencephaly in Cradd−/− Knockout Mice (A) Boxplot showing the ratio of endocranial volume to body weight in Cradd control (heterozygous and wild-type, Het-WT) (n = 9) and Cradd−/− knockout (Hom) (n = 8) mice. (B) Boxplot demonstrating the ratio of endocranial volume to humerus surface area in Cradd control (Het-WT) (n = 5) and Cradd−/− (Hom) (n = 3) mice. Wild-type and heterozygous mice are shown with closed and open circles, respectively.
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