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. 2018 Mar 1;141(3):651-661.
doi: 10.1093/brain/awx377.

A homozygous ATAD1 mutation impairs postsynaptic AMPA receptor trafficking and causes a lethal encephalopathy

Juliette Piard  1   2 , George K Essien Umanah  3   4 , Frederike L Harms  5 , Leire Abalde-Atristain  3   6 , Daniel Amram  7 , Melissa Chang  3   4 , Rong Chen  3   4 , Malik Alawi  8   9 , Vincenzo Salpietro  10 , Mark I Rees  11 , Seo-Kyung Chung  11 , Henry Houlden  10 , Alain Verloes  12 , Ted M Dawson  3   4   6   13   14 , Valina L Dawson  3   4   6   13   15 , Lionel Van Maldergem  1   2   16 , Kerstin Kutsche  5
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A homozygous ATAD1 mutation impairs postsynaptic AMPA receptor trafficking and causes a lethal encephalopathy

Juliette Piard et al. Brain. .

Abstract

Members of the AAA+ superfamily of ATPases are involved in the unfolding of proteins and disassembly of protein complexes and aggregates. ATAD1 encoding the ATPase family, AAA+ domain containing 1-protein Thorase plays an important role in the function and integrity of mitochondria and peroxisomes. Postsynaptically, Thorase controls the internalization of excitatory, glutamatergic AMPA receptors by disassembling complexes between the AMPA receptor-binding protein, GRIP1, and the AMPA receptor subunit GluA2. Using whole-exome sequencing, we identified a homozygous frameshift mutation in the last exon of ATAD1 [c.1070_1071delAT; p.(His357Argfs*15)] in three siblings who presented with a severe, lethal encephalopathy associated with stiffness and arthrogryposis. Biochemical and cellular analyses show that the C-terminal end of Thorase mutant gained a novel function that strongly impacts its oligomeric state, reduces stability or expression of a set of Golgi, peroxisomal and mitochondrial proteins and affects disassembly of GluA2 and Thorase oligomer complexes. Atad1-/- neurons expressing Thorase mutantHis357Argfs*15 display reduced amount of GluA2 at the cell surface suggesting that the Thorase mutant may inhibit the recycling back and/or reinsertion of AMPA receptors to the plasma membrane. Taken together, our molecular and functional analyses identify an activating ATAD1 mutation as a new cause of severe encephalopathy and congenital stiffness.

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Figures

Figure 1
Figure 1
DNA and RNA analysis in the family with three siblings carrying the homozygous ATAD1 mutation. (A) Pedigree of the family. (B) Partial sequence electropherograms demonstrating the ATAD1 c.1070_1071delAT [p.(His357Argfs*15)] mutation in the homozygous state in leucocyte-derived DNA of the affected siblings (Patients 1–3). Their healthy parents (father and mother) are heterozygous carriers of the mutation. (C) Partial sequence electropherograms show the 2-bp deletion in ATAD1 in fibroblast-derived cDNA of one sibling (Mutant) in comparison to the cDNA sequence of a healthy individual (Wild-type). Deleted bases are marked by parenthesis in the normal sequence. The encoded amino acid residues are depicted below each sequence in the three-letter code and show the 14 novel amino acid residues at the C-terminus of ATAD1 (highlighted in bold). Asterisk indicates a stop codon.
Figure 2
Figure 2
Mutant Thorase is expressed in patient-derived fibroblasts. (A) Immunoblot of lysates obtained from patient and control fibroblasts. Expression of Thorase was monitored by using anti-Thorase antibody, and anti-actin antibody was used to control for equal loading. As the anti-Thorase antibody was generated against the C-terminus and this region contains a new amino acid composition in the mutant, detection of Thorase in patient cells was difficult (compare the clear band in control and the diffuse band in patient cells). (B) Optical densitometry quantification of A. Values represent the mean ± SEM (n = 3, n.s. P > 0.05, Tukey’s multiple comparison tests).
Figure 3
Figure 3
The ATAD1 mutation p.(His357Argfs*15) leads to reduced amount of some mitochondrial proteins in patient-derived fibroblasts and locks Thorase in the oligomeric state. (A) Immunoblots of lysates obtained from patient and control fibroblasts. COX4 = cytochrome c oxidase subunit 4; HXK1, = hexokinase 1; PEX26 = peroxisomal biogenesis factor 26; VDAC1 = voltage dependent anion channel 1. (B) Optical densitometry quantification of (A). Values represent the mean + SEM (n = 3, ***P < 0.01, **P < 0.05, *P < 0.10, n.s. > 0.10, two-way ANOVA, Tukey’s multiple comparison tests). (C) Representative immunofluorescence images of the mitochondrial morphology (TOMM20 staining) in control and patient fibroblasts. The cells were also stained for Golgi (GOS28), peroxisomes (PEX26) and the nuclei with DAPI (blue). (D) Predicted 3D structure of Thorase wild-type (green) and mutantHis357Argfs*15 (red). (E) Size exclusive chromatograph profile of purified recombinant Thorase. Wild-type Thorase appears as a dimer (∼70 kDa), whereas the Thorase mutantHis357Argfs*15 appears as oligomer (>400 kDa). (F) Purified proteins resolved on 10% SDS-PAGE stained with coomassie (left) and immunoblotted with anti-Thorase antibody (right).
Figure 4
Figure 4
ATAD1 mutation p.(His357Argfs*15) affects GluA2-GRIP1 complex disassembly and GluA2 surface expression. (A) Immunoblot analyses of GST-Thorase pulldown of the GluA2-GRIP1 complex from Thorase knockout whole brain lysate in the presence of different nucleotides (ATP = hydrolysable ATP; ATPγS = non-hydrolysable ATP). The samples were incubated at 4°C for binding and then at 37°C for ATP hydrolysis to trigger the disassembly of the protein complex. (B and C) The graphs represent normalized per cent bound GluA2 (B) and GRIP1 (C) in the GST-Thorase pulldown samples for A. (D) Normalized percentage of GluA2 and GRIP1 disassembled from Thorase-GluA2-GRIP1 complex in A. (E and F) Representative immunofluorescence images of unstimulated and NMDA-induced endocytosis of GluA2 in Atad1−/− neurons expressing Thorase-GFP wild-type (WT) or the mutantHis357Argfs*15 (Mutant). (G) Normalized ratio of surface GluA2 (sGluA2) to internalized GluA2 (iGluA2) for E and F. (H) GluA2 internalization index measured as the ratio of iGluA2 to the total GluA2 (iGluA2 plus sGluA2) fluorescence intensities. (I) Immunoblot analyses of BS3-crosslinking of sGluA2 in Atad1−/− neurons expressing Thorase-GFP wild-type or mutantHis357Argfs*15. (J) The normalized optical densitometry quantification of sGluA2 for I. Mean ± SEM of three experiments performed in triplicate. n = 3, **P < 0.05, *P < 0.10, n.s. P > 0.10, ANOVA with Tukey-Kramer post hoc test when compared with wild-type.
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