ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

Abstract

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

Figure 1

Family with ATAD3A Walker A mutation. (A) Patient II-1 (35-year-old, female) has deformity in the lower extremities. (B) Magnetic resonance imaging of patient II-1 shows thinning and atrophy of the spinal cord (arrows). (C) Pedigree of the investigated family. Grandparents (I:1 and I:2) were not affected. Patient II:1 and her son III:1 have the heterozygous c.1064G > A variant in ATAD3A. (D) Sanger sequencing of the ATAD3A c.1064G > A variant in the samples of the family members I:2, II:1 and III:1. (E) Schematic presentation of the human ATAD3A protein and conservation of the Walker A motif. ATAD3 proteins contain two coiled-coil domains (Cc1 and Cc2), and an ATPase domain with Walker A (WA) and Walker B (WB) motifs. The glycine in the Walker A motif affected by the patient mutation G355D (arrow) is invariant in ATAD3 proteins between species, in human mitochondrial proteins with AAA modules and in the human ATAD family. The invariant lysine K358 is also indicated by an arrow. (F) In silico modeling of the G355D mutation suggests that the substitution of glycine 355 to aspartate changes the affinity of the substrate-binding site for ATP. Walker A motif is shown in red. (G) ATPase activities of the recombinant ATAD3A proteins (WT, patient mutant G355D and Walker dead K358A) assessed by the amount of phosphate liberated as described in the materials and methods. The measurements of five independent experiments are shown, the mean is indicated. The reactions were performed in the presence of 8 nM of ATAD3A protein. In the combined reactions of wild type and G355D mutant proteins 8 nM of each ATAD3A variant was used.

Figure 2

Overexpressed Walker A mutants induce mitochondrial fragmentation. (A) Fluorescent microscopy images of primary human fibroblasts transfected with mitochondria-targeted green fluorescent protein (mito-GFP) alone or together with wild type ATAD3A (WT), or ATAD3A Walker A mutants K358R or G355D. ATAD3A immunocytochemistry with ATAD3 antibody is shown. See Supplementary Material, Fig. S2 for merged images. (B) Western blotting shows equal levels of ATAD3A in cells transfected with WT, or the K358R or G355D mutant ATAD3A. GAPDH is shown as a loading control. (C) Quantification of the transfected cells displaying mitochondrial fragmentation in the experiment shown in A. Fragmented cells were manually counted four independent times in a randomized blinded manner (n = 157 transfected cells counted for each genotype). Means ± SEM are shown. *P < 0.05, **P < 0.005, one-way ANOVA with Bonferroni correction. (D) Merged images of fibroblasts transfected with WT, K358R or G355D ATAD3A plasmids, and stained with LysoTracker Green (lyso-Green) for lysosomes and MitoTracker Red (mito-Red) for mitochondria. Cells with fragmented mitochondrial networks show increased staining with LysoTracker Green. Scale bars = 20µm.

Figure 3

Mitochondrial elongation and upregulation of autophagy in patient fibroblasts. (A) Primary patient skin fibroblasts show an elongated mitochondrial morphology when compared with control cells. Mitochondria were immunolabeled with Tom20 (green) and ATAD3A (red) antibodies. (B) ATAD3A protein level is unchanged in patient (P) cells, but levels of Drp1 are reduced and the relative amounts of respiratory chain complex subunits are altered compared to controls (C) cells. (C) Live cell imaging shows an increase in the number of lysosomes (lyso-Green) and mitochondrial elongation (mito-Red) in patient neurons. Scale bars = 20µm. (D) Phosphorylation and levels of mTOR mediated basal autophagy markers ULK1 (Ser757) and S6 (Ser235/236) are altered in patient (P) cells compared to controls (C) under fed and starved conditions and when autophagy is inhibited (V, vehicle; B, bafilomycin) (E) Electron micrographs show large amounts of lysosomal structures (black arrow, upper panel) and unbranched elongated mitochondria (black arrow, lower panel) in the patient fibroblasts. (F) Lysosomal quantification by flow cytometry, (N_control =24459, N_patient =19517) ***P < 0.0001, Student’s t-test.

Figure 4

Increased lysosomes in patient-specific neurons. (A) Neurons derived through differentiation of ATAD3A patient’s induced pluripotent stem cells show altered appearance of the mitochondrial network and ATAD3 immunostaining compared to control neurons. Neurons were identified with TUJ1 (green), and labelled with ATAD3 antibody (red). The white arrows point to the neuronal soma and an extension, which are enlarged on the right. (B) Increased lysosomal structures are seen in patient neurons as stained with Lysotracker Green. (C) Patient (P) neuronal cultures have a comparable ATAD3A protein level to control (C) cells. (D) Electron micrographs show large amounts of lysosomal structures (black arrow) in the patient neurons. White arrows point to mitochondria. (E) The mitochondrial DNA copy number was comparable to control cells in the patient neuronal culture, as determined by quantitative PCR. NS, non-significant, Student’s t-test.