Mutations in APOPT1, encoding a mitochondrial protein, cause cavitating leukoencephalopathy with cytochrome c oxidase deficiency

Abstract

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

Figure 1

MRI Findings (A) MRI abnormalities observed in individual S6 in the acute stage at the age of 3 years. The sagittal image shows signal abnormalities in the posterior part of the corpus callosum and a single lesion at the genu (red arrows in A1). Axial T2-weighted (A2, red arrows), FLAIR (A3), and T1-weighted (A4) images show signal abnormalities predominantly involving the posterior part of the cerebral white matter and corpus callosum with numerous small and larger, well-delineated cysts. The diffusion-weighted images show that the noncavitated abnormalities have a high signal, suggesting diffusion restriction (red arrows in A5), as confirmed by the low signal on the apparent diffusion coefficient (ADC) maps (red arrows in A6). (B) MRI abnormalities observed in individual S4 in the subacute stage at the age of 5 years. The sagittal image shows the involvement of the posterior part of the corpus callosum (red arrow in B1). Axial T2-weighted (B2), FLAIR (B3), and T1-weighted (B4) images show signal abnormalities predominantly involving the posterior part of the cerebral white matter and corpus callosum with numerous small, well-delineated cysts. Additional minor abnormalities are seen next to the anterior horn of the lateral ventricle on the right (red arrows in B2 and B4). After contrast, enhancement of multiple foci is seen (red arrows in B4). The diffusion-weighted images show multiple small foci of high signal, suggesting diffusion restriction (red arrows in B5), as confirmed by the low signal on the ADC maps (red arrows in B6). Follow-up MRI of the same subject (B7–B10) shows striking improvement (B7 and B8). Involvement of long tracts within the brain stem is now visible (red arrows in B9 and B10). (C) Late follow-up MRI of individual S1 at age 21 shows atrophy and gliosis of what is remaining of the cerebral white matter (C1) with some small cysts in the abnormal white matter (C2). (D) MRI of individual S2 shows only minor posterior cerebral white matter abnormalities at age 15 (D1) with tiny cysts (D2).

Figure 2

Morphological Findings (A–C) The histochemical reaction to COX is diffusely decreased in muscle biopsies of individual S1 (A) and individual S2 (B), compared to a control biopsy (C). Scale bars represent 100 μm. (D) Electron microscopy of muscle from individual S1 shows abnormal mitochondria with osmiophilic inclusions and cristae disarray. Scale bar represents 0.3 μm. (E and F) Profound decrease of COX histochemical reaction is also visualized in fibroblasts from individual S6 (E) compared to a control cell line (F). Scale bars represent 10 μm.

Figure 3

APOPT1 Mutations and APOPT1 Localization (A–C) Mutations found in this study are positioned (arrows) against schematic representations of APOPT1 (A), cDNA (B), and protein (C). (D) The green fluorescence pattern of an APOPT1-GFP fusion protein starting from the methionine 14, transiently expressed in control fibroblasts (center), coincide with that obtained with mitotracker red (left), to give a yellow overlay pattern (right). Scale bar represents 10 μm.

Figure 4

Functional Studies of APOPT1 (A) Immunoblot analysis of fibroblasts stably expressing APOPT1-HA. No HA-immunoreactive material, visualized using a specific anti-HA antibody (α-HA, Roche), is present in naive conditions, whereas two HA-immunoreactive bands are detected under exposure of the cells to the proteasome inhibitor MG-132. Tubulin and SDHB, immunovisualized by specific antibodies (α-TUB, Sigma-Aldrich; α-SDH30, Mitoscience), are used as loading controls. (B) The anti-HA immunoreactive bands obtained as in (A) (arrows) have electrophoretic mobility identical to the in vitro translated cDNAs corresponding to the predicted precursor (prec) and mature (mat) APOPT1-HA protein species, synthesized using the TNT Transcription-Translation System kit (Promega). The in vitro translated products are specific, as no anti-HA immunoreactive material is visualized in the prereaction reticulocyte lysate (ret). Note that the panels are from the same filter, but different exposure times were used to better visualize the bands. (C) Anti-HA immunoreactive bands corresponding to the precursor and mature APOPT1 species are detected in fibroblasts exposed to H₂O₂. Note that the upper band, corresponding to the precursor APOPT1-HA species, is present in the sample collected 8 hr after the exposure to H₂O₂, whereas only the mature species is detected in samples collected at 24 and 48 hr, suggesting that over time the precursor APOPT1-HA species has been translocated across the inner mitochondrial membrane and quantitatively processed into the mature species by cleavage of a 39 amino acid N-terminal MTS. (D) ROS detection by dichlorofluorescein (DCHF, Invitrogen) fluorescence on control (Ct) and individual S2 fibroblasts in basal and oxidative stress conditions. Note that naive individual S2 fibroblasts show significantly higher levels of DCHF fluorescence under exposure to 100 μM and 1 mM H₂O₂. The levels of DCHF fluorescence are significantly lower in APOPT1-HA-expressing individual S2 fibroblasts. Bars represent standard deviations. The p values were obtained by unpaired, two-tail Student’s t test.

Figure 5

Complementation and RNAi Studies (A) Immunoblot analysis of one-dimension BNGE in immortalized fibroblasts from two controls (Ct1, Ct2) and mutant subjects S1 and S2. Samples were prepared using 10% w/v dodecylmaltoside. We used an antibody against NDUFA9 to detect complex I (I), an antibody against the subunit α of ATP synthase to detect complex V (V), an antibody against SDHA for complex II (II), an antibody against core I for complex III (III), and an antibody against subunit COX4 for complex IV (IV). Note that the amounts of cIV holocomplex and cIV+cIII2 supercomplex are clearly decreased in mutant samples compared to controls. (B) Immunoblot analysis of one-dimension BNGE in immortalized fibroblasts from a control (Ct), mutant S2, and S2 stably transduced with APOPT1-HA. Note that the amounts of cIV and cIV+cIII₂, which are markedly decreased in S2, are significantly increased in S2+APOPT1-HA. (C) Quantitative densitometric analysis of the results obtained by three independent BNGE experiments. The intensities of complex IV (CIV) and supercomplex III + IV (CIII+CIV) signals were normalized to complex II; the ratio obtained in control fibroblasts was set as 100%. The p values were obtained by unpaired, two-tail Student’s t test. Bars represent standard deviations. (D) Quantitative PCR analysis of APOPT1 transcript in naive, nontransduced immortalized fibroblasts, fibroblasts transduced with the “empty” vector pLKO.1, and with APOPT1-specific shRNA-2 and shRNA-3. The amount of APOPT1 transcript is decreased to approximately <5% by both shRNA, compared to the amount found in naive, nontransduced cells. (E) Growth curves in immortalized fibroblasts from a naive control cell line, and the same cell line transduced with empty vector (pLKO.1), shRNA-2, and shRNA-3. Cell lines with severe APOPT1 knockdown (down to around 1% of the control mRNA levels 48 hr postinfection) show significantly decreased cell growth.