Early-onset Parkinson disease caused by a mutation in CHCHD2 and mitochondrial dysfunction.

OBJECTIVE: Our goal was to identify the gene(s) associated with an early-onset form of Parkinson disease (PD) and the molecular defects associated with this mutation.
METHODS: We combined whole-exome sequencing and functional genomics to identify the genes associated with early-onset PD. We used fluorescence microscopy, cell, and mitochondrial biology measurements to identify the molecular defects resulting from the identified mutation.
RESULTS: Here, we report an association of a homozygous variant in CHCHD2, encoding coiled-coil-helix-coiled-coil-helix domain containing protein 2, a mitochondrial protein of unknown function, with an early-onset form of PD in a 26-year-old Caucasian woman. The CHCHD2 mutation in PD patient fibroblasts causes fragmentation of the mitochondrial reticular morphology and results in reduced oxidative phosphorylation at complex I and complex IV. Although patient cells could maintain a proton motive force, reactive oxygen species production was increased, which correlated with an increased metabolic rate.
CONCLUSIONS: Our findings implicate CHCHD2 in the pathogenesis of recessive early-onset PD, expanding the repertoire of mitochondrial proteins that play a direct role in this disease.

Parkinson disease (PD, MIM168600) is the most common movement disorder of aging and the second-most common neurodegenerative disease, after Alzheimer disease (MIM104300).[1,2] PD has been associated with mutations in multiple genes including PARK2, PINK1, PARK7, ATP13A2, SCNA, LRRK2, VPS35, EIF4G1, DNAJC13, and CHCHD2.[3-6]

Recent whole-exome sequencing (WES) of Japanese patients with autosomal dominant or sporadic PD identified a heterozygous CHCHD2 missense change (p.Thr61Ile) in a family with autosomal dominant late-onset PD.[5] Subsequently, the identical variant was identified in a Chinese family with autosomal dominant PD.[6] Additional CHCHD2 variants, including p.Ala32Thr, p.Pro34Leu, and p.Ile80Val, have been described in 4 western European familial patients with PD.[7] The pathomechanism of the CHCHD2 variants in these studies is, however, unclear as is the physiologic role of CHCHD2.

Here, we identify a new CHCHD2 variant in a patient with autosomal recessive early-onset PD and establish the pathogenic mechanism of this variant. We have shown that the mutation results in a fragmented mitochondrial morphology and reduced electron transport chain (ETC) activity.

Methods

Standard protocol approvals, registrations, and patient consents

The study was approved by the ethical standards of the relevant institutional review board, the Ethics Review Committee in the Gothenburg Region (Dn1: 842-14), and the Human Research Ethics Committee of the University of Western Australia. Informed consent was obtained from patients included in this study after appropriate genetic counseling. Blood samples were obtained from patients, their parents, and siblings.

Clinical evaluation

Medical history was obtained, and physical examination was performed as part of routine clinical workup.

Genetic analysis

Next-generation sequencing (NGS) whole-exome and/or targeted neuromuscular panel sequencing (WES or neuromuscular sub-exomic sequencing [NSES]) was performed on the patient's DNA. Confirmatory bidirectional Sanger sequencing was performed in patients with PD and their family members (see e-Methods, links.lww.com/NXG/A85).

Cell culture and transfections

Dermal fibroblast cultures from the patient were established by standard protocols, after written informed consent was obtained. Cultured fibroblasts from a healthy age-matched individual were used as a control. Detailed methods are provided in supplementary information.

Mitochondrial isolation and cell lysis

Mitochondria were isolated from fibroblasts as previously described.[8] Cell lysates were prepared using a buffer containing the following: 150 mM NaCl, 0.1% (vol/vol) Triton X-100, and 50 mM Tris-HCl (pH 8.0). Protein concentration was determined using a bicinchoninic acid (BCA) assay.

Fluorescence microscopy

Detailed methods are described in the supplementary material, links.lww.com/NXG/A85.

Long-range PCR and mitochondrial DNA copy number quantitative PCR

Detailed methods are described in the supplementary material. The sequences of all primers used for this study are detailed in table e-1, links.lww.com/NXG/A85.

SDS-PAGE and immunoblotting

Mitochondria (25 µg) isolated from control and patient fibroblasts were separated on 4%–12% Bis-Tris gels (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). All antibodies used are detailed in the supplementary information.

Mitochondrial protein synthesis

De novo mitochondrial protein synthesis was analyzed in fibroblasts using 35S-radiolabeling of mitochondrially encoded proteins in the presence of emetine as previously described.[9] The cells were suspended in phosphate-buffered saline (PBS) and 20 μg of protein was resolved on a 12.5% SDS-PAGE gel, and radiolabelled proteins were visualized on film.

Respiration

Respiration was measured in fibroblasts as previously described.[10] The full methods are detailed in supplementary information.

Cell function measurements

JC-1, mitochondrial mass, dihydroethidium (DHE), and MTS assay full methods are detailed in supplementary information.

Statistical analysis

All data are reported as mean ± SEM. Statistical differences were determined using a two-tailed Student t test. JC-1, DHE, mitochondrial mass, and MTS data are expressed as a percent of average control fibroblast values for the respective media treatment.

Data availability

Study data for the primary analyses presented in this report are available upon reasonable request from the corresponding and senior author.

Results

Clinical characteristics of the patient

A 30-year-old right-handed Caucasian woman (V:2) was born to healthy consanguineous parents aged 55 and 59 years (figure 1A). There was no significant family history, and she denied a history of other diseases or past drug use. The clinical presentations were consistent with PD. Symptoms developed rapidly at age 26 years including resting tremor mostly affecting her left arm and bradykinesia. Neurologic examination at age 28 years showed general bradykinesia, rigidity, resting tremor in both hands and legs, but most prominent in the left side, with superimposed action-induced myoclonus. She also showed hypomimic face and mydriatic pupils with sluggish reaction to the light, indicating autonomic dysfunction. There were no pyramidal or cerebellar signs. On fundoscopic examination, Kayser-Fleischer-like rings were negative. Neither sphincter dysfunction nor orthostatic hypotension was present. Laboratory evaluation including thyroid function, liver function, serum and urine copper, and ceruloplasmin was unremarkable. Brain MRI was also unremarkable. The patient showed several clinical features of mitochondrial dysfunction, such as neuropsychological disturbance, dysautonomia, and myoclonus. Low-dose levodopa was started with dramatic response, but after 1 month, she had severe dyskinesia, and after 3 months, motor fluctuation, anxiety, and insomnia became the main issues and she had dopamine dysregulation syndrome. After 6 months, psychiatric problems, including aggression, depression, and impulsiveness, were additional symptoms. At follow-up at age 30 years, PD symptoms had not progressed and became stable. However, she showed signs of cognitive decline, and orthostatic hypotention added to her symptoms.

Figure 1

Pedigree and genetic findings

(A) Pedigree of the family. The affected individual (V:2) is represented with a shaded symbol. (B) Sanger sequence analysis demonstrates the presence of homozygous variants in CHCHD2 and (C) TOP1MT in the patient and the segregation of the variants. (D) Multiple sequence alignment confirms that the p.Ala71Pro (CHCHD2) and p.Asp221Asn (TOP1MT) substitutions affect evolutionarily conserved residues (shaded). (E) Mutated residues and CHCHD2 protein structure: previously identified heterozygous missense variants associated with familial PD (black arrows) and the currently identified homozygous variant (red arrow) are all located in exon 2. The CHCHD2 p.Ala71Pro amino acid substitution is located within the central conserved hydrophobic domain at the transmembrane element.

Genetic findings

Data from NGS of DNA from 1 affected (V:2) and 1 unaffected family member (V:1) were analyzed. No likely pathogenic mutations were identified in the genes included in the targeted panel of the 336 neuromuscular disease genes. WES was performed on the patient and her unaffected sister. No rare, likely pathogenic heterozygous variants in the PD-associated genes were identified. The filtering strategy of initially concentrating on homozygous coding variants in known neurogenetic disease genes, selected based on variant databases Human Genome Mutation Database and ClinVar, and the most recent literature allowed the identification of homozygous CHCHD2 (chromosome 7) and TOP1MT (chromosome 8) variants. A novel homozygous missense mutation in exon 2 of CHCHD2 (c.211G>C) (p.Ala71Pro) was identified. The size of the homozygous region covering CHCHD2 variant on chromosome 7 was 158Mb. In addition, a homozygous missense variant of TOP1MT (c.661G>A, ENST00000523676, transcript ID: NM_001258447.1), changing aspartic acid to asparagine (p.Asp221Asn), was identified. No rare, likely pathogenic heterozygous or homozygous variants in the PD-associated genes, including ATP13A2, CHCHD10, DNAJC13, EIF4G1, LRRK2, PARK2, PARK7, PARK15, PINK1, SNCA, and VPS35, were identified in the exome sequencing data. The appearance of CHCHD2 and TOP1MT variants was examined in available family members by sequencing analysis. Sanger sequencing confirmed segregation of both variants compatible with a recessive pattern of inheritance. The unaffected parents (IV:1, IV:2) were both heterozygous for the CHCHD2 variant, and the healthy sister (V:1) was not a carrier (figure 1B). Both parents and the sister were heterozygous carriers of the TOP1MT variant (figure 1C). The c.211G>C, (p.Ala71Pro) (CHCHD2) variant was excluded in East and South Asian, European, African, Latino, and Ashkenazi Jewish Allele Frequency Communities (AFC), in the ExAC database, the Genome Aggregation Database (gnomAD), and the 1000 Genome database. The TOP1MT variant (c.661G>A, p.Asp221Asn, rs143769145) was a rare heterozygous variant reported in the AFC (frequency 0.025%), the ExAC (frequency 0.026%), and in the gnomAD (frequency 0.023%) databases; the variant was identified in the homozygous state. In silico combined annotation dependent depletion (CADD) analysis of the missense variants revealed high deleterious scores (CHCHD2 [p.Ala71Pro]: 32.000 and TOP1MT [p.Asp221Asn]: 26.000). In silico analysis predicted both CHCHD2 and TOP1MT substitutions to be potentially disease causing (MutationTaster, mutationtaster.org/). The 2 amino acid residues affected are highly conserved across species (figure 1D). The CHCHD2-substituted residue is located within the central conserved hydrophobic domain, the transmembrane element of the CHCHD2 protein (figure 1E). In addition, the entire coding sequence of CHCHD2 was analyzed in 2 affected individuals of 2 large families with familial early-onset PD of Iranian origin. The CHCHD2 p.Ala71Pro substitution was not identified, and we did not detect any other variant in this gene.

Clinical status of confirmed carriers

Cosegregation studies confirmed that the parents were carriers of CHCHD2 and TOP1MT variants, and the unaffected sister was heterozygous for the TOP1MT variant. Given that the previously reported CHCHD2 variants are mostly associated with late-onset dominant PD, carrier parents were examined by a neurologist. They showed no evidence of neurologic or movement disorder by history. However, the parents are still younger than the average age of PD onset (<60).[11]

Reduction of CHCHD2 but not TOP1MT causes the PD pathology

To investigate the molecular mechanisms behind this form of early-onset PD, dermal fibroblasts were established from the patient and compared with controls established from a healthy individual. We investigated the protein abundance by immunoblotting in patient and control fibroblasts to determine which variant was pathogenic. We observed a small reduction in TOP1MT levels, whereas the CHCHD2 reduction was more pronounced (figure 2A). As TOP1MT is the topoisomerase that catalyzes supercolied mtDNA relaxation,[12] we investigated mtDNA stability and abundance. Long-range PCR showed no mtDNA fragmentation and changes in mtDNA size in patient cells (figure 2B). Furthermore, qPCR analysis showed no significant difference in mtDNA copy number, indicating that the TOP1MT variant has negligible effects on mtDNA structure (figure 2, B and C).

Figure 2

CHCHD2 but not TOP1MT contributes to PD pathogenesis

(A) SDS-PAGE and immunoblotting were performed on 25 μg of isolated mitochondria from control and patient fibroblasts. (B) Mitochondrial DNA integrity was examined using long-range PCR of control and patient fibroblast DNA. DNA was separated on a 1% agarose gel stained with ethidium bromide. (C) Mitochondrial DNA copy number was examined using quantitative PCR of control and fibroblast DNA. (D) Mitochondrial morphology of control and patient fibroblasts grown in either high glucose or galactose media was examined using MitoTracker Orange and visualized by fluorescence microscopy. The scale bar represents 10 µm. Arrows indicate refractile granules characteristic of network fragmentation. (E) Twenty-five micrograms of isolated mitochondria from control and patient fibroblasts was analyzed for mitochondrial contact site and cristae organizing system (MICOS) protein levels by immunoblotting. SDHA was used as a loading control.

CHCHD2 has been shown to regulate mitochondrial morphology in Drosophila melanogaster.[13] Therefore, we investigated mitochondrial morphology in patient cells using MitoTracker Orange. In glucose medium, we identified differences in morphology and fusion between patient and control cells, characterized by a reduced reticular network, fragmentation of filamentous mitochondria, and accumulation of granular bodies around the nucleus (figure 2D). We cultured both fibroblasts in galactose medium that stimulates a dependence on aerobic metabolism compared with cells grown in glucose media, which can rely on anaerobic metabolism. Growth in galactose exacerbated differences in mitochondrial morphology seen in patient cells (figure 2D). Our experiments excluded the mtDNA abnormalities that would be linked to TOP1MT dysfunction and identified changes in mitochondrial morphology that is related to the proposed function and membrane association identified for CHCHD2 as summarized in figure e-1, links.lww.com/NXG/A85.

The role of other CHCHD family proteins in the mitochondrial contact site and cristae organizing system (MICOS) complex[14,15] lead us to investigate how reduced CHCHD2 expression affects MICOS complex subunits. Although levels of the intermembrane protein CHCHD3 were reduced in patient cells, levels of the lipid binding proteins APOO and APOOL were unchanged (figure 2E). We conclude that although CHCHD2 does not have a direct role in MICOS complex function or stability, the CHCHD2 variant results in reduction of the membrane-associated protein CHCHD3.

Reduced CHCHD2 expression results in OXPHOS dysfunction

Impaired oxidative phosphorylation (OXPHOS) complex formation and function has been identified in PD and drug-induced parkinsonism.[16] Therefore, we investigated OXPHOS complex subunit levels by immunoblotting. We found reduced levels of complex I, IV, and V subunits, whereas complexes II and III showed negligible reductions in patient cells (figure 3A). De novo mitochondrial translation examined using 35S-methionine showed no difference in mitochondrial translation rates between control and patient cells (figure 3B). This indicates that the CHCHD2 mutation causes changes in OXPHOS subunit abundance through reduced protein stability and morphologic changes, not impaired protein synthesis.

Figure 3

Patient cells show impaired OXPHOS complex levels and function

(A) SDS-PAGE and immunoblotting were performed on 25 μg of isolated mitochondria from control and patient fibroblasts. (B) Mitochondrial protein synthesis was examined in control and patient fibroblasts using 35S radiolabeling of mitochondrial translation products. Twenty micrograms of cell lysate was separated on a 12.5% SDS-PAGE gel and visualized by autoradiography. (C) Oxygen consumption of specific respiratory complexes was measured in control and patient fibroblasts using an OROBOROS respirometer. (D) Phosphorylated (state 3) and nonphosphorylated (state 4) respiration was measured in the presence of 10 mM succinate/0.5 μM rotenone in digitonin-permeabilized control and patient cells to determine the respiratory control ratio under both glucose and galactose media conditions. Data are presented as mean ± SEM and were analyzed using a Student's t test. *p < 0.05, **p < 0.01.

Next, we investigated ETC activity by measuring mitochondrial respiration at each complex (figure 3C). We show reduced oxygen consumption at complexes I and IV but not complexes II and III, suggesting that CHCHD2 may regulate electron transfer between these complexes, as previously suggested.[13,17] We also examined the respiratory control ratio (RCR) by measuring oxygen consumption with succinate and rotenone under ATP-generating conditions (phosphorylating state 3) and non-ATP generating conditions (nonphosphorylating state 4). We identified significant decreases in the RCR in patient cells grown in glucose and galactose, which is characteristic of an uncoupling of the ETC and ATP production (figure 3D). This indicates that reductions in OXPHOS activity are likely due to impaired mitochondrial morphology and electron transport between complexes.

To test whether CHCHD2 dysfunction resulted in PD, we performed rescue experiments by expressing the wild-type CHCHD2 or TOP1MT proteins in the control and patient cells (figure 4). The fragmented mitochondrial network was restored to filamentous, reticular appearance (figure 4A), and respiration was restored to levels comparable to the control fibroblast when rescued by expression of the wild-type CHCHD2 protein, but not by expression of the wild-type TOP1MT protein (figure 4B). These experiments validated the causative role of the CHCHD2 mutation in PD pathology.

Figure 4

CHCHD2 but not TOP1MT expression rescues molecular defects

(A) Mitochondrial morphology of control and patient fibroblasts, in the presence or absence of either wild-type CHCHD2 or wild-type TOP1MT, was examined using MitoTracker Orange and visualized by fluorescence microscopy. The scale bar represents 10 µm. Arrows indicate refractile granules characteristic of network fragmentation. (B) Oxygen consumption of respiratory complexes was measured in control, patient fibroblasts, and patient fibroblasts expressing either wild-type CHCHD2 or wild-type TOP1MT using an OROBOROS respirometer. Data are presented as mean ± SEM and were analyzed using a Student's t test. *p < 0.05, **p < 0.01.

Mutation in CHCHD2 causes increased oxidative stress

Next, we investigated the mitochondrial membrane potential (Δψm) using JC-1. There were no significant changes in the Δψm in patient cells grown in glucose or galactose, indicating that the proton motive force maintains the Δψm (figure 5A). The decrease in membrane potential was greater in patient cells treated with FCCP, indicating a reduction in maximal respiratory capacity. This reduction was greater under galactose conditions (figure 5, A and B). The reduced stability of OXPHOS subunits and decreased respiratory capacity were independent of mitochondrial mass (figure 5C).

Figure 5

Patient cells demonstrate increased oxidative stress

(A) JC-1 assay was used to examine the strength of the proton motive force in both control and patient fibroblasts under high glucose (A) and galactose (B) media conditions both in the absence of FCCP (+Δψm) and the presence of FCCP (−Δψm) (n = 6). (C) Mitochondrial mass was measured in control and patient fibroblasts using nonyl acridine orange (NAO) under high glucose conditions (n = 6). (D) Reactive oxygen species (ROS) levels were measured in control and patient fibroblasts using dihydroethidium (DHE) under both high glucose and galactose media conditions (n = 6). (E) Metabolic activity was measured in control and patient fibroblasts using an MTS assay (n = 6). All data are presented as mean ± SEM and were analyzed using a Student's t test. *p < 0.05, ***p < 0.001.

Next, we investigated reactive oxygen species (ROS) production.[18] Patient cells showed significantly increased superoxide levels when grown in glucose, which was further exacerbated when cells were grown in galactose (figure 5D). As ROS have been shown to affect metabolic rate and viability, we investigated the metabolic consequences of increased ROS levels. Patient cells showed a mild metabolic increase under glucose conditions and a further increase under galactose conditions, indicating that the increase in ROS is not inherently cytotoxic, but is sufficient to modulate the metabolic rate in skin fibroblasts (figure 5E).

Discussion

Here, we have identified homozygous CHCHD2 and TOP1MT variants in a Caucasian woman presenting with characteristic features of PD at 26 years. Both healthy parents were heterozygous CHCHD2 and TOP1MT variant carriers, but not the unaffected sister. The likely pathogenicity of the variants was supported by the results of the prediction tools PolyPhen-2, SIFT, and CADD, where the TOP1MT variant (c.661G>A, p.Asp221Asn, rs143769145) was identified at a low frequency of 0.023%–0.025% in the Genome Aggregation Databases with no homozygotes. The mutated residues of both CHCHD2 and TOP1MT are highly conserved during evolution. Taken together, our finding suggests that both CHCHD2 and TOP1MT might be causative genes of recessive early-onset PD. Mitochondrial dysfunction has been shown to be a key factor in PD pathogenesis in both animal models and patients, with disease-associated genes PINK1 and CHCHD2 being involved in mitochondrial function.[13,19-21] Recently, however, the association of CHCHD2 with PD has displayed mixed results in different populations,[6,7,19,22-25] suggesting that it is infrequent and ethnic specific. Here, we have used cultured patient fibroblasts to establish the pathogenic mechanisms of the identified CHCHD2 and TOP1MT variants and expand the repertoire of genes implicated in the pathology of PD.

Homozygous variants of both CHCHD2 and TOP1MT affected protein levels, albeit more pronounced for CHCHD2. In patient fibroblasts, the reduced CHCHD2 expression caused mitochondrial network fragmentation when cells were forced to rely on oxidative phosphorylation. This is consistent with previous studies that implicated CHCHD2 in mitochondrial morphology and its importance for energy production.[13,26] The morphology and respiration defects in the patient fibroblasts were rescued by expressing wild-type CHCHD2, providing evidence that the CHCHD2 mutation contributes to the PD pathology. Reduced TOP1MT expression causes increased negative supercoiling of mtDNA, resulting in reduced mtDNA replication.[26] However, the lack of changes in mtDNA integrity or copy number in patient fibroblasts indicated that the TOP1MT variant had a negligible effect on TOP1MT function. Furthermore, the contribution of the TOP1MT variant to the PD pathogenesis was excluded because expression of this protein did not rescue mitochondrial morphology and function.

Changes in mitochondrial morphology have been previously linked with PD-characteristic OXPHOS deficiencies.[16,20,21,27,28] Reductions in complexes I and IV subunits were consistent with reduced activities of these complexes in patient cells. Although reduced complex IV activity has been previously reported in PD, there is much less evidence for impaired complex IV activity, contributing to PD development.[27] The reduction in complex IV formation is consistent with studies finding an interaction between CHCHD2 and the cytochrome c–binding protein MICS1.[13,29] The reduction in complex activity and function was consistent with changes in the mitochondrial membrane and indicated an uncoupling of the ETC and impaired electron transfer through the ETC,[30,31] as seen previously.[17] It is possible that reduced CHCHD2 expression causes impaired MICS1 function, therefore impairing movement of electrons from complex III to IV by cytochrome c, as previously suggested.[17]

The Δψm was not significantly altered in patient fibroblasts despite inefficient electron transfer and reduced respiratory capacity. The greater difference in uncoupled state shown under galactose conditions indicates that when patient cells are made to rely on aerobic energy sources, they can compensate for abnormalities in ETC coupling and the electron leak through increasing the electrons flowing through the OXPHOS complexes. Although increasing electron flow may allow cells to maintain the proton motive force, it has negative consequences through ROS formation.

The increased ROS levels were not cytotoxic and, in contrast, patient fibroblasts demonstrated increased metabolic rate. Although mild increases in ROS levels have been shown to increase metabolic rate and proliferation in certain cells, such as fibroblasts, neuronal cells show reduced viability.[32-35] The mitochondrial toxicity of oxidized dopamine derivatives, in combination with the inherently low mitochondrial mass of DA neurons, may potentiate mitochondrial dysfunction resulting from reduced CHCHD2 expression and cause neuronal cell death.[33] As dermal fibroblasts may not fully emulate the characteristics of DA neurons, further investigation is needed to understand how increased ROS levels alter metabolic function in DA neurons and how they potentiate mitochondrial dysfunction resulting from reduced CHCHD2 expression.

Recent findings demonstrate that mutations in CHCHD2 and its paralogue CHCHD10 are associated with multiple neurodegenerative disorders.[25,36-38] The existence of a complex containing CHCHD2 and CHCHD10 may explain shared features between disorders.[39,40] In addition, CHCHD10 knockdown and knockout models show altered respiratory activity and OXPHOS subunit levels.[39,40] Further investigation is required to fully understand how this complex regulates mitochondrial and neurologic functions.