Abstract
Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson’s disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as ‘seeds’ and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.
Introduction
Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurones and the formation of intracellular Lewy bodies (LBs), which consist primarily of |$a$|-synuclein (1). Interactions between genetic predisposition and environmental factors are likely to be primarily responsible for mitochondrial dysfunction and oxidative damage, which in turn drive oligomerization and aggregation of synuclein; however, the underlying molecular mechanisms remain poorly understood. The vast majority of PD is sporadic, and inherited familial forms of the disease accounting for only 5% of all cases (2). The identification of PD-related genes and risk factors has implicated several pathways in PD etiology, with accumulating evidence suggesting a link between dysfunctional intracellular protein catabolism, mitochondrial clearance and PD pathogenesis. In PARK1-linked PD, intrinsically disordered mutant synuclein initiates the disease process. Given that highly aggregated proteins are deposited in nigral neurones in PD, dysfunctions of proteolytic systems, i.e. the ubiquitin–proteasome system and autophagy–lysosomal pathway, are likely to contribute to the final neurodegenerative process. In this context, the stress-inducible protein p62 (encoded by the gene Sqstm1) is important because it is an ubiquitin- and LC3-binding protein that functions as a selective autophagy adaptor/receptor for degradation of ubiquitinated substrates (3). Indeed, p62 is present in neuronal inclusions of individuals with Alzheimer’s disease and other neurodegenerative diseases (4), although its actual roles in pathogenesis remain largely unknown.
In a previous study by our group, linkage analysis and whole-genome sequencing of Japanese families with an autosomal dominant form of PD identified mutations in CHCHD2 (5). Patients with these variants manifested typical late-onset, levodopa-responsive parkinsonism with a benign course. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates PD pathology. Autopsy of an individual with the T61I mutation revealed widespread |$a$|-synuclein accumulation (6). CHCHD2 localizes to the intermembrane spaces of the mitochondria and plays roles in the regulation of mitochondrial respiration, transcription of complex IV and mitochondria-associated apoptosis (7,8). Loss of CHCHD2 destabilizes cytochrome c and complex IV in cultured cells and causes degeneration of mitochondrial cristae and impairment of oxygen respiration, leading to loss of dopaminergic neurones and motor dysfunction (9). To elucidate the critical role of CHCHD2 in dopaminergic (DA) neurones, we characterized the age-related pathological and motor phenotypes in CHCHD2-deficient mice.
Results
To determine the function of CHCHD2 in vivo, we generated Chchd2-knockout (KO) mice. A targeting vector was constructed using 1.5- and 7-kb DNA fragments as the 5′ and 3′ homologous sequences, respectively. A negative selection cassette, DTA, which encodes the diphtheria toxin, was also included (Fig. 1A). The linearized targeting vector was transfected into C57BL/6 embryonic stem (ES) cells. After selection in G418, clones were screened for homologous recombination by Southern blotting. Using the 5′ and 3′ external probe (Fig. 1B) and a probe specific for the neo sequence (data not shown), we confirmed that the clones carried the desired homologous recombination. ES cells derived from these clones were injected into C57BL/6 embryos, and chimeric offspring were crossed with C57BL/6 mice to obtain germline. Heterozygous mice were then interbred to obtain homozygous KO and wild-type control mice. Mice were subsequently genotyped by PCR using primers specific for the wild-type and targeted alleles (data not shown). KO mice were completely defective in CHCHD2 expression (Fig. 1C and D). And CHCHD2 is mainly expressed in the heart, liver and brain (Fig. 1E). To identify the localization of endogenous CHCHD2, we performed double immunostaining with antibodies with CHCHD2 and cytochrome C in the heart, liver and dopaminergic cells. Endogenous CHCHD2 is ubiquitously localized in the mitochondria (Supplementary Material, Fig. S1).
Generation of CHCHD2-deficient mice. (A) Schematic representation of the targeting vector and the targeted allele of the Chchd2 gene. The 5.8-kb region of the mouse Chchd2 gene, including exons 1–3, was followed by an FRT-flanked PGK-neo expression cassette in the opposite transcriptional orientation. In the targeting construct, a 5.2-kb 5′ fragment and a 2.4-kb 3′ fragment were used as the long and short homologous arms, respectively. (B) Southern blot analysis of genomic DNA from ES cells that had undergone homologous recombination. Genomic DNA was digested with EcoRI/ApaI and hybridized with the 5′ probe or 3′ probe. Left panel: The 5′ probe detected a 16.3-kb band from the wild-type allele and 10.3-kb band from the targeted allele. Right panel: The 3′ probe detected a 6.0-kb band from the wild-type allele and 8.7-kb band from the targeted allele. (B) Expression of CHCHD2 transcript in cortex, striatum, and midbrain, as determined by RT-PCR. The amplified region was between exon 3 and exon 11. Actin cDNA was amplified as an internal control. (D) Immunoblot of CHCHD2 in mouse brain. Mitochondrial fractions of whole brain from the indicated genotypes were immunoblotted for CHCHD2 and HSP60. (E) Expression levels of CHCHD2 in the indicated organs of mice. Lysates of whole tissue of the indicated genotypes were immunoblotted for CHCHD2 and GAPDH. CHCHD2 was expressed at the highest levels in heart and liver.
Initially, we characterized the locomotor impairments of CHCHD2 KO mice. KO mice were viable at birth, indistinguishable in appearance from their littermates, and had a normal survival rate. Although these mice have not yet been observed over their entire lifespan, they began to exhibit motor behavioral deficits around 115 weeks. These clinical abnormalities could be observed in the runway test (Fig. 1Aa, Ab and B, and Supplementary Material, Video S1, CHCHD2 KO mouse; Supplementary Material, Video S2, CHCHD2 wild-type mouse) and rotarod tests (Fig. 1C). In the runway test, wild-type mice exhibited well-coordinated movement and almost no slips of the forepaw or hindpaw from the beam; by contrast, CHCHD2 KO mice could hardly move on the beam, and frequently slipped. In particular, the hindpaws of CHCHD2 KO mice often slipped off the beam (Fig. 1Ab and B). Furthermore, in the accelerating rotarod test, fall latency was reduced in CHCHD2 KO mice (Fig. 1C). There were no effects on lifespan (Fig. 2D) or weight (Fig. 2E).
Behavioral examinations of aged CHCHD2 KO mice. (A) Runway test of wild-type mice (a) and CHCHD2 KO mice (b). In contrast to wild-type mice, which exhibited well-coordinated movement and almost no slips of the forepaw or hindpaw from the beam, CHCHD2 KO mice could hardly move on the beam, and their hindpaws slipped frequently (see Supplementary Material, Video S1, CHCHD2 KO; Supplementary Material, Video S2, CHCHD2 wild-type). These experiments were performed using mice over 110 weeks of age. In this figure, the hindpaw of the Parkin KO mouse has slipped off the beam. (B) The number of hindlimb slips was recorded from 80-week-old (CHCHD2 wild-type, n = 10; CHCHD2 KO, n = 10) and 120-week-old (CHCHD2 wild-type, n = 10; CHCHD2 KO, n = 10) mice crossing the challenge beam. Data are means ± SE (error bars); statistical significance was evaluated using Student’s t-test. **P < 0.01. (C) In the accelerating rotarod assay, rotation was accelerated from 3 to 40 rpm over 5 min, and fall latency was recorded. The experiments were performed using mice from 80 to 125 weeks of age (CHCHD2 wild-type, n = 10; CHCHD2 KO, n = 10). Red line: CHCHD2 KO mice; blue line: wild-type mice. Data are means ± SE (error bars); statistical significance was evaluated using Student’s t-test. **P < 0.01. (D) Kaplan–Meier analysis of survival of CHCHD2 KO (n = 20) and wild-type (n = 20) mice. Data are presented as means ± SE (error bars); **P < 0.01 (ANOVA, Tukey post hoc pairwise comparisons). Red line: CHCHD2 KO mice; blue line: wild-type mice. (E) Body weight of mice from 90- to 120-week-old mice (CHCHD2 KO mice, n = 20; wild-type mice, n = 20).
Previous studies showed that loss of autophagy in the central nervous system (CNS) leads to formation of inclusions positive for the autophagy adaptor/receptor protein p62 in various neuronal populations (3). In addition, we reported that loss of Atg7 leads to age-related development of p62 inclusions in dopaminergic (DA) neurones; on the basis of this finding, we suggested that p62 aggregates that form due to autophagic defects might serve as ‘seeds’ for Lewy body (LB) formation (10). However, the mechanisms underlying age-related progression and intracellular localization of these inclusions in dopaminergic neurones remain elusive.
Next, we investigated the p62 pathology of the substantia nigra (SN) in CHCHD2 KO mice. Initially, we confirmed aggregate formation by immunohistochemical staining. Age-dependent p62-positive inclusions were specifically observed in DA neurones in the SN (Fig. 3A and C). And none of p62 aggregates were observed in other CHCHD2-positive tissues such as liver, heart and kidney in aged mice (Supplementary Material, Fig. S2). In contrast to younger CHCHD2 KO mice, in which no aggregates were seen even at the age of 12 months (data not shown), in 120-week-old mice, p62 inclusions were present in DA neuron cell bodies and outside the soma (Fig. 3Aa and Ab). To determine the location of these inclusions, in a previous study, we conducted double staining with p62 and TH antibodies and showed that p62 inclusions were present along the fibers, mainly in the branch of dopaminergic neurones (10). We assumed a similar pathology in CHCHD2-deficient mice. In electronic microscope study, we actually confirmed fibrous or amorphous structure within the fibers of SN (Fig. 3B).
Development of p62-positive aggregates in dopaminergic neurones. (A) Histological analyses of DA neurones in 120-week-old CHCHD2 KO (a–c) and wild-type (d–f) mice. Cryosections were immunostained for p62 or synuclein. In contrast to wild-type mice, in which no aggregates were seen in dopaminergic neurones, the abundance of p62- (arrow) or synuclein-positive aggregates increased in the dopaminergic neurones of CHCHD2 KO mice. Scale bars: (a), (d) 50 μm; (c), (d) 20 μm. High magnification of p62 aggregates (a) is shown (b). High magnification of synuclein aggregates (c) is shown in the inset. Scale bar: (b), (e) 20 μm. (B) Electron micrographs in the substantia nigra (SN). Fibrous (a–c) and amorphous (d–f) structures accumulate within neural fibers. Square area in (a) is enlarged in (b). Square area in (b) is enlarged in (c). Square area in (d) is enlarged in (e). Square area in (e) is enlarged in (f). Scale bars: (a) 5 μm; (b), (d) 1 μm; (c), (f) 100 nm; (e) 500 nm. (C) p62 aggregates colocalized with synuclein. Immunofluorescence labeling of synuclein (red) or p62 (green) in the substantia nigra of CHCHD2 KO mice. (a) Low-magnification immunofluorescence labeling in the SN. (b–d) Higher magnification of p62 and synuclein-positive neurones is shown in the inset. Scale bars: 20 μm.
Previous in vivo analyses of DA neurones suggested that synuclein regulation is linked to autophagy (11,12), but it remained unclear how synuclein degradation is regulated. To address this issue, we investigated synuclein homeostasis and colocalization with p62-positive inclusions in DA neurone-specific Atg7-deficient mice (10). To this end, we pathologically confirmed endogenous synuclein accumulation and colocalization with p62 inclusions. High-resolution confocal images through the SN following immunofluorescence labeling of synuclein (Fig. 3Cb) and p62 (Fig. 3Cc) revealed that synuclein was located in p62-positive aggregates (Fig. 3Cd). Interestingly, these inclusions were present not only within the soma, but also outside the soma.
Although p62 is a representative substrate of autophagy that is rapidly influenced by its dysfunction, it seemed that not only p62, but also endogenous synuclein, was regulated to some extent by autophagy. Thus, loss of CHCHD2 might be associated with autophagy dysfunction and formation of LBs. Accumulating knowledge regarding CHCHD2, which is associated with mitochondria, has increased our understanding of the protein’s cellular functions. In fact, CHCHD2 is associated with mitochondrial respiration (5), cytochrome c oxidase expression (13), complex IV activity (7) and inhibition of apoptosis (8) in vitro; however, it remains unclear how CHCHD2 influences autophagy, mitochondrial structure or mitochondrial function in vivo.
To further characterize the mitochondria, we performed ultrastructural analysis in DA neurones of 120-week-old mice (Fig. 4Aa–c; wild-type control, Fig. 4Ad–f; CHCHD2 KO mouse sample). We specifically observed small, round, fragmented mitochondria in CHCHD2-deficient DA neurones (Fig. 4Ad–f), but not in controls (CHCHD2 wild-type) (Fig. 4Aa–c). Precise quantification revealed that mitochondrial area was reduced in CHCHD2-deficient DA neurones (Fig. 4Ak). Furthermore, fragmented mitochondria had normal outer membrane structure, but exhibited an irregular inner structure (Fig. 4Ae and Af), i.e. the normal structures of matrix and cristae were broken. As in Parkin KO mice, mitochondria with damaged internal structure accumulated in CHCHD2 KO mice. In Parkin KO mice, we observed small, round, fragmented mitochondria in DA neurones, leading us to speculate that damaged mitochondria accumulate due to abrogation of PINK1/Parkin-mediated mitophagy.
We next asked whether CHCHD2-mediated mitophagy contributes to mitochondrial degradation or affects the fusion/fission system. In order for damaged mitochondria to be degraded by autophagy, they must be segregated by fission (14). Thus, CHCHD2 KO mice may have damaged mitochondria. Indeed, the activities of complexes I and III were reduced in the brains of CHCHD2 KO mice brain (Fig. 4B).
Accumulation of damaged mitochondria in CHCHD2-deficient mice. (A) Electron micrographs of DA neurones in the substantia nigra (SN), cortex (g, h) and striatum (i, j). (a–c, g, i) 120-week-old wild-type mice. Square area in (a) is enlarged in (b). Square area in (b) is enlarged in (c). (d–f, h, i) 120-week-old CHCHD2 KO mice. Square area in (d) is enlarged in (e) and (f). Fragmented mitochondria with damaged inner structure accumulate in the CHCHD2 KO mice. Scale bars: (a), (d) 2 μm; (b), (e), (g-j) 1 μm; (c), (f) 500 nm. (k) Quantification of mitochondrial area (wild-type DA cell, n = 20; CHCHD2 KO DA cell, n = 20). In CHCHD2-deficient DA neurones, mean mitochondrial area was smaller than in wild-type mice. Data are means ± SE (error bars); statistical significance was evaluated using Student’s t-test. **P < 0.01. (B) Quantification of mitochondria complex activity in the brain (wild-type, n = 6; CHCHD2 KO, n = 6). Activities of complex I and complex III were reduced in CHCHD2-deficient mice. (C) Dysfunction OPA1 proteolysis in brain mitochondria was accelerated in CHCHD2-deficient mice (wild-type, n = 3; CHCHD2 KO, n = 3). (a) Ratio of long to short form of OPA1. (b) Ratio of long form density to total density. (c) Band density of total Drp1. (d) S616 Drp1/total Drp1 ratio. (e) S637 Drp1/total Drp1 ratio. Data are means ± SE (error bars); statistical significance was evaluated using Student’s t-test. *P < 0.05.
Mitochondria are highly dynamic organelles that are critical for energy production and numerous metabolic processes. Liu et al. (15) showed that CHCHD2 regulates mitochondrial morphology and tissue homeostasis by controlling the levels of OPA1, a large GTPase that regulates the fusion of inner mitochondria membranes (16). Hence, we measured in vivo OPA1, Drp1, phosphorylated Drp1 (Fig. 4C) and Mfn1/2 (Supplementary Material, Fig. S5) levels. Interestingly, the ratio of the long and short forms of OPA1 was reduced in brain mitochondria (Fig. 4C). Specifically, the long form of OPA1 was spliced or degraded in the brain mitochondria. On the other hand, Drp1, phosphorylated Drp1 (Fig. 4C) and Mfn1/2 (Supplementary Material, Fig. S5) levels did not differ in CHCHD2 KO mice. Studies using mammalian cells have shown that OPA1 can be processed by YME1L and OMA1 (17–19). In CHCHD2-deficient mice, the mitochondrial protein profile in the brain may be damaged.
CHCHD10 is a paralogous protein of CHCHD2 and cause amyotrophic lateral sclerosis. CHCHD2 and CHCHD10 are redundant and form heterodimers (20). We performed western blotting for CHCHD10 in brain and liver tissues (Supplementary Material, Fig. S6). We have detected the decrease of CHCHD10 levels in CHCHD2-knockout tissues. CHCHD2 may contribute to the stability of CHCHD10. The differences in the stability and mutual affinity of CHCHD2 and CHCHD10 regulate their heterodimerization (20).
Although the molecular components required for mitophagy have been identified through extensive in vitro work, the physiological pathology and context of these pathways remain largely unknown (21). To assess the contribution of accumulation of damaged mitochondria, we compared the number of TH neurones between aged CHCHD2 KO and control mice. As demonstrated in the rotarod test, CHCHD2 KO mice exhibited locomotor dysfunction at ages above 115 weeks (Fig. 2C). We sacrificed 120-week-old mice (CHCHD2 KO, n = 5; wild-type, n = 5) and counted TH neurones in three sections (VTA: ventral tegmental area; SNcc: center area of substantia nigra pars compacta; SNcl: lateral area of substantia nigra pars compacta) (Fig. 5A). CHCHD2 KO mice had fewer TH neurones than wild-type control mice. The reduction in TH cell number was most prominent in SNcc (Fig. 5B). We conducted stereological quantification of the area of cell bodies, but we did not observe a difference between CHCHD2 wild-type and CHCHD2 KO mice. Consistent with our results, a reduction in the abundance of TH neurones also occurs in PD, primarily in the SNcc. Loss of DA neurones may contribute to the motor impairment observed in the late stages of disease. In addition, we tested DA physiology in these 120-week-old CHCHD2 KO mice by neurochemical analysis of the dorsal striata. High-performance liquid chromatography (HPLC) revealed a reduction in striatal DA levels in CHCHD2 KO mice relative to control mice (Fig. 5C). Thus, DA content is affected by loss of DA neurones. Intriguingly, the significant decrease in dopamine was not accompanied by concomitant reductions in the dopamine catabolites DOPAC and HVA. Currently, we have not clarified the reason for this, but it is possible that abnormal mitochondrial accumulation may induce partially modified DA metabolism (22).
Delayed degeneration of DA neurones in CHCHD2 KO mice. (A) Histological analyses of SN in 120-week-old CHCHD2 KO and wild-type mice. Paraffin sections were immunostained for TH. SNcl: lateral area of substantia nigra pars compacta; SNcc: center area of substantia nigra pars compacta; VTA: ventral tegmental area. Scale bars: 20 μm. (B) DA neurones in the SN were identified by TH immunoreactivity. Data are means ± SE (CHCHD2 KO mice, n = 5; wild-type mice, n = 5); **P < 0.01 (Student’s t-test). (C) HPLC analysis of DA (left), DOPAC (middle), and HVA (right) levels in dorsal striatum at 120 weeks. Data are means ± SE (CHCHD2 KO, n = 5; wild mice, n = 5); *P < 0.05 (Student’s t-test).
Discussion
We demonstrated dopaminergic-specific mitochondrial dysfunction including function and structure, and inclusion formation in the CHCHD2-deficient mice. What regulates mitochondrial damage and/or inclusion bodies formation? We evaluated mitochondrial DNA copy number normalized by nuclear DNA for each tissue (23). The number of mitochondria DNA tends to decrease in the midbrain of CHCHD2-deficient mice. On the other hand, mitochondrial DNA copy number tends to increase in the liver and kidney (Supplementary Material, Fig. S7), which are CHCHD2-positive tissues other than the brain. CHCHD2 deficiency may not affect mitochondrial DNA copy number.
To identify the cause of inclusion formation, we investigated mitochondrial complex activity (Supplementary Material, Fig. S4) and mitochondrial structure (Supplementary Material, Fig. S3) in the liver, which is a typical CHCHD2-positive tissue. In the liver, only complex I activity was decreased, but no mitochondrial structural abnormality was observed. On the other hand, in the brain, decreased activity of complexes I and III and structural abnormalities of mitochondria were observed. Because complexes I and II complement their functions, it may be difficult to appear the phenotype as a damage mitochondrial function in liver. Then, to facilitate mitochondria fragmentation, it must be required to reduce the complex activity to some extent.
Interestingly, mitochondria dysfunction and structural changes (fragmentation) are brain-specific features. Especially, these changes were found in the substantia nigra (Fig. 4Ak). Regardless of the endogenous expression level of CHCHD2, it may have protective function to maintain mitochondrial structure in the cells. While we performed western blotting for LC3 to evaluate the activity of autophagy (Supplementary Material, Fig. S8). We detected no difference in the amount of LC3-II between wild-type and CHCHD2 KO mice. Thus, we suppose that the deficiency of CHCHD2 does not affect the activity of autophagy.
Genetic studies using mice have highlighted the importance of constitutive autophagy in nondividing cells such as neurones, in which loss of autophagy causes cytoplasmic inclusions formation and neurodegeneration (24,25). Komatsu et al. reported that p62/A170/SQSTM1 plays critical roles in the formation of protein aggregations and is removed by autophagy (3). In fact, DA neuron–specific autophagy-deficient mice have demonstrated in vivo evidence for LB formation. Namely, synuclein deposition is preceded by p62 and resulted in the formation of inclusions containing synuclein and p62 (10).
A variety of PD models have been generated to study different aspect of PD for understanding the pathogenesis. Genetic model is a compelling approach to examine the association of specific mutations in the familial forms of PD. However, few of these models reproduce the complete features of the disease. Most of the genetic models failed to induce significant loss of dopaminergic neurones and the main hallmark of PD (26). As to mitochondria-associated genes including Parkin and PINK1, none of the knockout models recapitulate typical phenotype of PD. CHCHD2-deficient mice, which show dopaminergic neuronal loss and Lewy body-like inclusion formation, may provide a useful platform to study the pathophysiology of PD. From this standpoint, the observation that CHCHD2 KO mice both exhibit a PD-related phenotype and harbor p62-positive inclusion represents a breakthrough. Although the molecular components required for autophagy have been identified through extensive in vitro work, the relationship between CHCHD2 and these pathways remains unknown. DA neurones derived from iPSC clones from a patient with the CHCHD2 T61I mutation contained elevated levels of the membrane-bound form of LC3B (LC3B-II), suggesting that autophagic flux was likely to be activated (6). In the normal state, CHCHD2 is not colocalized in LBs (27); furthermore, when mitochondria are damaged, CHCHD2 accumulates in the mitochondria and facilitates CHCHD10 oligomerization (20). However, CHCHD10 did not undergo oligomerization in the CHCHD2 KO mice brain (Supplementary Material, Fig. S6). Even if mitochondrial membrane potential has not decreased sufficiently in the CHCHD2 KO mice, it remains unclear why Parkin KO mice, which are assumed to have damaged mitochondria, do not accumulate p62-positive aggregates (28). From this standpoint, it appears that the change in mitochondrial membrane potential is unrelated.
In this study, we showed that a lack of CHCHD2 is associated with inclusion body formation, even in the absence of proteolytic dysfunction. Therefore, we predict that mitochondrial function will play an even more critical role in the pathogenesis of PD when disease-related aggregation-prone proteins are expressed as a result of genetic mutations or environmental insults, leading to neurodegeneration.
Materials and Methods
Animals
All animals were kept in a pathogen- and odor-free environment, which was maintained under a 12-h light/dark cycle at ambient temperature. Procedures were approved by the Animal Experimental Committee of the Juntendo University Graduate School of Medicine and were performed in accordance with the guidelines of NIH and the Juntendo University Graduate School of Medicine.
Behavioural tests
Locomotor behavior was assessed in mice from 80 to 125 weeks of age. Accelerating rotarod tests were performed on a rotarod machine with automatic timers and falling sensors (MK-660D; Muromachi Kikai). CHCHD2 KO and wild-type mice (n = 10 for each genotype) were placed on a 3-cm diameter rotating rod covered with rubber, and rotation was accelerated from 3 to 35 rpm over 5 min. Fall latency was recorded, and the first fall latency of the third trial was used for analysis. The runway test was performed using a narrow, horizontally fixed beam (1 cm wide, 80 cm long, held at a height of 40 cm from the table). The animal was placed at one end of the beam and urged to move toward the opposite end, where an escape platform was located.
Immunoblot analyses
Anaesthetized mice were dissected by decapitation, and lysates for SDS-PAGE were prepared from tissues including brain as described previously (29). After electrophoresis, separated proteins were transferred to PVDF membranes (Hybond-P; Amersham Biosciences), which were incubated overnight at 4°C with the indicated primary antibodies: anti-CHCHD2, anti-ATP5B, anti-HSP60 (ab46798; Abcam), anti-GAPDH (10494–1-AP; Proteintech), anti-OPA1 (clone18; BD), or anti-Drp1 (611 113; BD), anti-Drp1(S616) (#4494; Cell Signaling), anti-Drp1(S637) (#6319; Cell Signaling), anti-Mfn1 (H0005569; Abnova), anti-Mfn2 (sc-100 560; Santa Cruz), anti-TIM23 (611 222, BD), anti-CHCHD10 (HPA003440; Atlas Antibodies), anti-actin (MAB1501; Millipore), anti-p62 (PM045; MBL), anti-LC3 (5F10; nanotools, anti-tubulin (T9026; Sigma-Aldrich). Antibody binding was visualized using the Enhanced Chemiluminescence Kit (GE Healthcare), and signals were detected on an ImageQuant LAS4000 (GE Healthcare).
Histological analyses
Mice were perfused with 4% paraformaldehyde (PFA), and their brains were immersion fixed at 4°C for 36 h. The fixed samples were cryoprotected with 20% sucrose and sliced on a freezing microtome to obtain 40-μm-thick floating sections. For double immunofluorescence study of synuclein (red) and p62 (green), floating sections were incubated with the rabbit anti-synuclein antibody (AB5038P; Millipore) and guinea pig anti-p62 antibody (PROGEN Biotechnik, GmbH). The sections were then incubated with Alexa Fluor 546-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-guinea pig IgG. Fluorescent signals were captured on an LSM 780 confocal microscope (Zeiss).
Mitochondrial isolation and assays for activity of mitochondrial complexes
Mouse brains were homogenized with a glass-Teflon Potter homogenizer in 0.3 M mannitol solution containing 10 mM HEPES (pH 7.4), 0.2 mM EDTA and 0.1% BSA. Homogenates were centrifuged at 740 × g for 10 min to remove unbroken cells and nuclei. Mitochondria were obtained by centrifugation of supernatants at 3000 × g for 8 min and at 7600 × g for 5 min, and then the mitochondria were washed twice and suspended in 0.3 M mannitol solution containing 10 mM HEPES (pH 7.4) and 0.1% BSA. For detection of complex I activity, mitochondria (5 μg, three freeze–thaw cycles) were suspended in 30 mM potassium phosphate buffer (pH 7.5) containing 1 mM MgCl2, 70 μM coenzyme Q1 (Sigma), 2.6 mM KCN, 67 μM antimycin A, 0.67% BSA and 0.2 mM NADH with or without 60 μM rotenone. Activity was analyzed as a decrease in absorbance of NADH at 340 nm. For detection of complex II activity, the isolated mitochondria (1.5 μg, one freeze–thaw cycle) were added to 50 mM potassium phosphate buffer (pH 7.5) containing 100 μM coenzyme Q1, 2 mM KCN, 0.1 mM 2,6-dichloroindophenol sodium salt hydrate (DCPIP) (Sigma) and 10 mM succinate with or without 10 mM malonic acid. Activity was analyzed as a decrease in the absorbance of DCPIP at 600 nm. For detection of complex III activity, the isolated mitochondria (1.5 μg, one freeze–thaw cycle) were added to 30 mM potassium phosphate buffer (pH 7.5) containing 1 mM MgCl2, 2.6 mM KCN, 2.5% BSA, 83 μM rotenone, 90 μM Ubiquinol (Carbosynth) and 50 μM oxidized cytochrome c with or without 70 μM antimycin A. Activity was analyzed as an increase in absorbance of reduced cytochrome c at 550 nm. For detection of complex IV activity, isolated mitochondria (0.5 μg, one freeze–thaw cycle) were added to 50 mM potassium phosphate buffer (pH 7.5) containing 66 μM reduced cytochrome c (Cayman 700 992) with or without 2 mM KCN. Activity was analyzed as a decrease in the absorbance of reduced cytochrome c at 550 nm.
Electron microscopy
For conventional electron microscopy, mice were fixed by cardiac perfusion with 2.5% glutaraldehyde in 0.1 mol/L PB (pH 7.2). Brain slices were embedded in epoxy resin, and ultrathin sections (70 nm thick) were cut and observed on an HT7700 electron microscope (Hitachi). Using these electron microscopy data, the number and area of mitochondria within dopaminergic neurones were manually measured using the cellSens analytical software (Olympus).
HPLC analysis
Dorsal striata from 120-week-old mice were dissected, quickly frozen on dry ice, and then homogenized with 0.5 mL of 0.2 M perchloric acid containing 100 μM EDTA-2Na per 100 mg wet tissue. Samples were centrifuged at 20 000 × g for 15 min at 4°C. The supernatant was collected and analyzed by HPLC.
Stereology quantification
For stereological quantification, three areas (VTA, the ventral tegmental area; SNcc, center area of the substantia nigra pars compacta; SNcl, lateral area of the substantia nigra pars compacta) were selected. For each genotype, every other 40-μm section of serial coronal brain slices was stained for TH (657 012; Calbiochem). Quantification was performed with design-based stereology system (Stereo-Investigator version 2019; MBF Bioscience). Sampling parameters were set up according to the software guide to achieve a coefficient of error between 0.06 and 0.09 using the Gundersen test, normally with a counting frame size 30 × 30 μm, and optical dissector an average of 10 sampling sites per section.
Statistical analysis
Statistical significance was determined by Student’s t-test (STATVIEW; SAS Institute) or ANOVA with Tukey post hoc pairwise comparisons. Data are expressed as means ± S.E. P < 0.05 was considered significant.
Abbreviations
PD: Parkinson’s disease, LBs: Lewy bodies, DA: dopaminergic, KO: knockout, ES: embryonic stem, SN: substantia nigra, HPLC: high-performance liquid chromatography, DCPIP: 2,6-dichloroindophenol sodium salt hydrate, VTA: the ventral tegmental area, SNcc: center area of the substantia nigra pars compacta, SNcl: lateral area of the substantia nigra pars compacta
Acknowledgements
We thank Dr Souichiro Kakuta (Juntendo University) for excellent technical assistance with electron microscopy and Dr Takashi Ueno (Juntendo University) for providing ATP5B antibody.
Conflict of Interest statement. The authors declare no competing interests.
Funding
This work was supported by KAKENHI (NH, #19 K22603; SS, #19 K07850; SN, #19 K23782) from JSPS and Comprehensive Brain Science Network (CBSN) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
