Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disorder, characterized by progressive cerebral demyelination cerebral childhood adrenoleukodystrophy (CCALD) or spinal cord neurodegeneration (adrenomyeloneuropathy, AMN), adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFA) in tissues. The disease is caused by mutations in the ABCD1 gene, which encodes a peroxisomal transporter that plays a role in the import of VLCFA or VLCFA-CoA into peroxisomes. The Abcd1 knockout mice develop a spinal cord disease that mimics AMN in adult patients, with late onset at 20 months of age. The mechanisms underlying cerebral demyelination or axonal degeneration in spinal cord are unknown. Here, we present evidence by gas chromatography/mass spectrometry that malonaldehyde-lysine, a consequence of lipoxidative damage to proteins, accumulates in the spinal cord of Abcd1 knockout mice as early as 3.5 months of age. At 12 months, Abcd1- mice accumulate additional proteins modified by oxidative damage arising from metal-catalyzed oxidation and glycoxidation/lipoxidation. While we show that VLCFA excess activates enzymatic antioxidant defenses at the protein expression levels, both in neural tissue, in ex vivo organotypic spinal cord slices from Abcd1- mice, and in human ALD fibroblasts, we also demonstrate that the loss of Abcd1 gene function hampers oxidative stress homeostasis. We find that the alpha-tocopherol analog Trolox is able to reverse oxidative lesions in vitro, thus providing therapeutic hope. These results pave the way for the identification of therapeutic targets that could reverse the deregulated response to oxidative stress in X-ALD.     

A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily. The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation. It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes. Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA. The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype. These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.     

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies.     

X-linked adrenoleukodystrophy: role of very long-chain acyl-CoA synthetases

The principal biochemical abnormality in the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is elevated plasma and tissue levels of very long-chain fatty acids (VLCFA). Enzymes with very long-chain acyl-CoA synthetase (VLACS) activity are required for VLCFA metabolism, including degradation by peroxisomal beta-oxidation or incorporation into complex lipids, and may also participate in VLCFA synthesis. Two enzymes with VLACS activity, ACSVL1 and BG1, were investigated for their potential role in X-ALD biochemical pathology. Skin fibroblast mRNA levels for ACSVL1, an enzyme previously shown to be in peroxisomes and to participate in VLCFA beta-oxidation, were not significantly different between normal controls, patients with childhood cerebral X-ALD, and patients with adrenomyeloneuropathy. Similar results were obtained with mRNA for BG1, a non-peroxisomal enzyme that is highly expressed in nervous system, adrenal gland, and testis, the principal tissues pathologically affected in X-ALD. No significant differences in the immunohistochemical staining patterns of tissues expressing either ACSVL1 or BG1 were observed when wild-type and X-ALD mice were compared. Western blot analysis of BG1 protein levels showed no differences between fibroblasts from controls, cerebral X-ALD, or adrenomyeloneuropathy patients. BG1 protein levels were similar in wild-type and X-ALD mouse brain, spinal cord, testis, and adrenal gland. We hypothesized that one function of BG1 was to direct VLCFA into the cholesterol ester synthesis pathway. However, BG1 depletion in Neuro2a cells using RNA interference did not decrease incorporation of labeled VLCFA into cholesterol esters. We conclude that the role, if any, of ACSVL1 and BG1 in X-ALD biochemical pathology is indirect.     

X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in elevated levels of very long-chain fatty acids (VLCFA) and reduced VLCFA beta-oxidation in peroxisomes. The peroxisomal membrane harbors three additional closely related ABC half-transporters, ALDRP, PMP70 and PMP69 (PMP70R). ABC half-transporters must dimerize to form a functional full-transporter. Whether ALDP forms a homodimer or a heterodimer has not yet been resolved, but most indirect evidence favors homodimerization. The peroxisomal ABC half-transporters are functionally related. Over-expression of ALDRP can correct the biochemical defect both in X-ALD patients cells and the Abcd1 knockout mouse, providing an exciting new possibility for treatment of X-ALD patients. This paper provides an overview of current knowledge and the problems that have been encountered.     

Toxic effects of X-linked adrenoleukodystrophy-associated, very long chain fatty acids on glial cells and neurons from rat hippocampus in culture

Saturated very long chain fatty acids (VLCFAs; > or =C22:0) accumulate in X-linked adrenoleukodystrophy (X-ALD, OMIM 300100), a severe hereditary neurodegenerative disease, due to peroxisomal impairment. Previous studies analysed the development of X-ALD in humans and gene knockout animal models. However, the toxic effect of VLCFA leading to severe symptoms with progressive and multifocal demyelination, adrenal insufficiency and inflammation still remains unclear. To understand the toxic effects of VLCFA in the brain, here we exposed neural cells to VLCFA and analysed the cellular consequences. We found that oligodendrocytes and astrocytes challenged with docosanoic- (C22:0), tetracosanoic- (C24:0) and hexacosanoic acids (C24:0) die within 24 h. VLCFA-induced depolarization of mitochondria in situ and increased intracellular Ca2+ level in all three brain cell types provides indications about the mechanism of toxicity of VLCFA. Interestingly, VLCFAs affect to the largest degree the myelin-producing oligodendrocytes. In isolated mitochondria, VLCFAs exert a detrimental effect by affecting the inner mitochondrial membrane and promoting the permeability transition. In conclusion, we suggest that there is a potent toxic activity of VLCFA due to dramatic cell physiological effects with mitochondrial dysfunction and Ca2+ deregulation. This provides the first evidence for mitochondrial-based cell death mechanisms in neurodegenerative disease with peroxisomal defects and subsequent VLCFA accumulation.     

Evaluation of the therapeutic potential of PPARalpha agonists for X-linked adrenoleukodystrophy

Adrenoleukodystrophy protein (ABCD1), a peroxisomal membrane protein, is mutated in patients affected by X-linked adrenoleukodystrophy (X-ALD). Adrenoleukodystrophy-related protein (ABCD2) is the closest relative of ABCD1. Pharmacological induction of ABCD2 gene expression has been proposed as a novel therapy strategy for X-ALD. Fibrates induce peroxisome proliferation and Abcd2 expression in rodent liver. Here we evaluate the possibility of using peroxisome proliferator-activated receptor alpha (PPARalpha) agonists for pharmacological induction of ABCD2 expression. In the liver of PPARalpha-deficient mice, both the constitutive and the fenofibrate-inducible Abcd2 gene expression was found to be PPARalpha-dependent. In the brain, PPARalpha-deficiency has no effect on Abcd2 expression. In mice orally treated with the novel, highly selective, and potent PPARalpha agonists GW 7647, GW 6867, and tetradecylthioacetic acid, Abcd2 expression was induced in liver and adrenal glands, but not in brain and testis. None of four putative PPREs identified in the 5(')-flanking DNA and in intron 1 of the Abcd2 gene conferred fibrate response in luciferase reporter assays. Thus, although fibrate-mediated Abcd2 induction is PPARalpha-dependent, it appears to be an indirect mechanism. Within the mouse Abcd2 promoter, a putative sterol regulatory element (SRE) similar in sequence and position to the characterized SRE sequence of the human ABCD2 promoter, was identified. A PPARalpha dependent induction of the sterol regulatory-binding protein 2 (SREBP2) and a down-regulation of SREBP1c mRNA levels could be demonstrated after fenofibrate treatment of mice. Our results suggest that the PPARalpha agonist-mediated induction of Abcd2 expression seems to be indirect and possibly mediated by SREBP2.     

Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders.     

Determination of 30 X-linked adrenoleukodystrophy mutations, including 15 not previously described

X-linked Adrenoleukodystrophy (X-ALD) is the most frequent peroxisomal disease. It mainly involves the nervous system white matter, adrenal cortex and testes. Several distinct clinical phenotypes are known. The principal biochemical abnormality is the accumulation of saturated very-long-chain fatty acids (VLCFAs : > C22:0, mainly C26:0), which is due to impaired capacity for beta-oxidation in peroxisomes. Diagnosis is usually based on the VLCFA levels in plasma or cultured skin fibroblasts in both patients and carriers. In 0.1% of affected males, however, the plasma C26:0 level is borderline normal, and 15% of obligate female carriers have normal results. Effective mutation detection in these families is therefore fundamental to unambiguously determine the genetic status of each individual at risk. Of particular concern are female members of kindreds segregating X-ALD mutations, because normal VLCFA levels do not guarantee lack of carrier status. We describe a fast method for detection of X-ALD mutations. The method is based on SSCP analysis of nested PCR fragments followed by sequence-determination reactions. Using this methodology we have found X-ALD mutations in 30 kindreds, including 15 not previously reported.     

Peroxisomal very long chain fatty acid beta-oxidation activity is determined by the level of adrenodeukodystrophy protein (ALDP) expression

Impaired peroxisomal beta-oxidation of saturated very long chain fatty acids (VLCFA, >/=C22:0) results in increased VLCFA levels in the tissues and body fluids of patients with disorders of peroxisomal biogenesis (i.e., Zellweger syndrome and neonatal adrenoleukodystrophy) and single peroxisomal protein defects (i.e., X-linked adrenoleukodystrophy (X-ALD) and acyl-CoA oxidase deficiency). We show that SV40T transformation also results in impaired peroxisomal beta-oxidation and VLCFA accumulation despite the presence of abundant peroxisomes. To explore the mechanism responsible for this observation, we have examined expression of key components of peroxisomal VLCFA beta-oxidation. We found that expression of both acyl-CoA oxidase, the rate limiting enzyme of peroxisomal VLCFA beta-oxidation and the adrenoleukodystrophy protein (ALDP), the defective gene product in X-ALD, are reduced after SV40T transformation. Surprisingly, ALDP overexpression by itself restores peroxisomal VLCFA beta-oxidation in SV40T-transformed control and X-ALD cells. These results demonstrate that ALDP is a fundamental component in VLCFA peroxisomal beta-oxidation and may serve as a "gatekeeper" for VLCFA homeostasis.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Photoreceptor cell apoptosis in the retinal degeneration of Uchl3-deficient mice

UCH-L3 belongs to the ubiquitin C-terminal hydrolase family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. A murine Uchl3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. To elucidate the function of UCH-L3, we investigated histopathological changes and expression of apoptosis- and oxidative stress-related proteins during retinal degeneration. In the normal retina, UCH-L3 was enriched in the photoreceptor inner segment that contains abundant mitochondria. Although the retina of Uchl3-deficient mice showed no significant morphological abnormalities during retinal development, prominent retinal degeneration became manifested after 3 weeks of age associated with photoreceptor cell apoptosis. Ultrastructurally, a decreased area of mitochondrial cristae and vacuolar changes were observed in the degenerated inner segment. Increased immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment indicated mitochondrial oxidative stress. Expression of cytochrome c, caspase-1, and cleaved caspase-3 did not differ between wild-type and mutant mice; however, immunoreactivity for endonuclease G was found in the photoreceptor nuclei in the mutant retina. Hence, loss of UCH-L3 leads to mitochondrial oxidative stress-related photoreceptor cell apoptosis in a caspase-independent manner. Thus, Uchl3-deficient mice represent a model for adult-onset retinal degeneration associated with mitochondrial impairment.     

Caveolin-1(-/-)- and caveolin-2(-/-)-deficient mice both display numerous skeletal muscle abnormalities, with tubular aggregate formation

Here, we examine the role of "non-muscle" caveolins (Cav-1 and Cav-2) in skeletal muscle biology. Our results indicate that skeletal muscle fibers from male Cav-1(-/-) and Cav-2(-/-) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells. Notably, these skeletal muscle defects were more pronounced with increasing age. Because Cav-2-deficient mice displayed normal expression levels of Cav-1, whereas Cav-1-null mice exhibited an almost complete deficiency in Cav-2, these skeletal muscle abnormalities seem to be due to loss of Cav-2. Thus, Cav-2(-/-) mice represent a novel animal model-and the first genetically well-defined mouse model-that can be used to study the pathogenesis of tubular aggregate formation, which remains a poorly understood age-related skeletal muscle abnormality. Finally, because Cav-1 and Cav-2 were not expressed within mature skeletal myofibers, our results indicate that development of these abnormalities probably originates in stem/precursor cells, such as satellite cells or myoblasts. Consistent with this hypothesis, skeletal muscle isolated from male Cav-3(-/-) mice did not show any of these abnormalities. As such, this is the first study linking stem cells with the genesis of these intriguing muscle defects.     

Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction

MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity.     

Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.     

Diagnostic yield muscle biopsy in patients with clinical evidence of mitochondrial cytopathy

We retrospectively reviewed 118 muscle biopsy specimens from 113 patients with clinical and/or biochemical evidence of mitochondrial cytopathy. Light microscopic evaluation revealed histologic abnormalities in 65 specimens. The most common histologic findings included angular atrophic esterase-positive muscle fibers, type II muscle atrophy, regenerating muscle fibers, and scattered cytochrome-oxidase deficient fibers. Ragged red fibers were noted in 3 specimens on a Gomori trichrome stain. Electron microscopic evaluation was performed in 113 muscle specimens, and in 34, no abnormalities were identified. Increased numbers of mitochondria, particularly in the subsarcolemmal region, were identified in 54 specimens. Increased mitochondrial size was seen in 8 specimens and paracrystalline mitochondrial inclusions in 3. Other ultrastructural findings included focally increased glycogen deposition, focal Z-band streaming, and focally increased lipid accumulation. For 39 cases, concomitant skin biopsy specimens were available; abnormalities were identified by electron microscopy in 12. The majority of biopsy specimens demonstrated some light or electron microscopic abnormality. Specific histologic findings suggestive of mitochondrial abnormalities (partial cytochrome oxidase deficiency, ragged red fibers) were noted in a minority of cases. Ultrastructural evidence of mitochondrial abnormalities was noted in the majority of cases.     

Cerebral inflammation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disease that affects approximately 1 in 25 000 males. It is characterized by elevated levels of saturated very long chain fatty acids (VLCFA), i.e., >C22:0, particularly in ganglioside and cholesterol ester fractions of brain white matter and adrenal cortex. Failure of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) to activate these VLCFA prevents their degradation by peroxisomal beta-oxidation. X-ALD maps to Xq28 and the gene encodes a peroxisomal membrane protein and not the gene for VLCS. The two most common forms of X-ALD are the cerebral (CER) form, with an inflammatory demyelinating reaction that resembles multiple sclerosis (MS), and adrenomyeloneuropathy (AMN), which involves the spinal cord and in which the inflammatory reaction is mild or absent. Investigations into the nature of the cerebral inflammatory demyelinating reaction in X-ALD will be the subject of this review.     

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.