Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses

Mitochondrion 2 (2002) 143–147 www.elsevier.com/locate/mito

Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses Ramon Marti a, Antonella Spinazzola a, Ichizo Nishino a, Antonio L. Andreu a, Ali Naini a, Saba Tadesse a, Juan A. Oliver b, Michio Hirano a,* a

Department of Neurology, Columbia University College of Physicians & Surgeons, P&S 4-443, 630 West 168th Street, 10032 New York, NY, USA b Department of Medicine, Columbia University College of Physicians & Surgeons, New York, NY, USA Received 4 December 2001; accepted 27 February 2002

Abstract Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease with mitochondrial DNA (mtDNA) alterations and is caused by mutations in the nuclear gene encoding thymidine phosphorylase (TP). The cardinal clinical manifestations are ptosis, ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Skeletal muscle shows mitochondrial abnormalities, including ragged-red fibers and cytochrome c oxidase deficiency, together with mtDNA depletion, multiple deletions or both. In MNGIE patients, TP mutations cause a loss-of-function of the cytosolic enzyme, TP. As a direct consequence of the TP defect, thymidine metabolism is altered. High blood levels of this nucleoside are likely to lead to mtDNA defects even in cells that do not express TP, such as skeletal muscle. We hypothesize that high concentrations of thymidine affect dNTP (deoxyribonucleoside triphosphate) metabolism in mitochondria more than in cytosol or nuclei, because mitochondrial dNTPs depend mainly on the thymidine salvage pathway, whereas nuclear dNTPs depend mostly on de novo pathway. The imbalance in the mitochondrial dNTP homeostasis affects mtDNA replication, leading to mitochondrial dysfunction. q 2002 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved. Keywords: Mitochondria; Mitochondrial DNA; Thymidine phosphorylase; Multiple deletions; Depletion; Nucleotide pool

1. Introduction Mitochondrial encephalomyopathies encompass a diverse group of diseases caused by dysfunction of the mitochondrial respiratory chain. Mitochondrial enzymes that form the respiratory chain are encoded by two genomes, nuclear DNA (nDNA) and mito-

* Corresponding author. Tel.: 11-212-305-1048; fax: 11-212305-3986. E-mail address: [email protected] (M. Hirano).

chondrial DNA (mtDNA). As a result, mitochondrial encephalomyopathies can be caused by defects in either genome or in the intercommunication between the genomes. During the last 13 years, investigators have focused research on mtDNA leading to the identification of more than 100 distinct point mutations, as well as large-scale molecular rearrangements (Servidei, 2001). Since 1995, several mutations of nDNA have been identified as causes of respiratory chain monoenzymopathies (Bourgeron et al., 1995; DiMauro, 1999). Until 1999, however, there were no known mutations of nDNA leading to depletion or multiple deletions of mtDNA.

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2. Genetic origin of MNGIE We mapped the MNGIE locus to the chromosome 22q13.32-qter (Hirano et al., 1998), and identified thymidine phosphorylase (TP) as the causative gene for the disease (Nishino et al., 1999). We have identified more than 20 different mutations in MNGIE patients, including missense, splice-site, microdeletions and one single nucleotide insertion. All of the patients were found to be either homozygotes for one mutation or compound heterozygotes, consistent with the autosomal recessive inheritance of the disease.

Fig. 1.

3. TP function and consquences of TP mutations

Mitochondrial neurogastrointestinal encephalomyopathy (MNGlE) is an autosomal recessive disease with alterations in mtDNA (Hirano et al., 1994; Papadimitriou et al., 1998). The clinical features that define MNGIE are: (1) gastrointestinal dysmoltility; (2) cachexia; (3) ptosis and progressive external ophthalmoplegia; (4) peripheral neuropathy; (5) leukoencephalopathy, and (6) laboratory evidence of mitochondrial dysfunction. Mitochondrial dysfunction in skeletal muscle in MNGIE has been reported in several studies (Bardosi et al., 1987; Hirano et al., 1994; Papadimitriou et al., 1998) and includes ragged-red fibers with ultrastructurally abnormal mitochondria, decreased activities of respiratory chain enzymes, and multiple mtDNA deletions or mtDNA depletion, or both.

TP enzyme, also known as platelet-derived endothelial cell growth factor (Usuki et al., 1992), is a homodimer (Kubilus et al., 1978; Miyazono and Heldin, 1989) that catalyzes the reversible phosphorolysis of thymidine, yielding thymine and 2-deoxyribose 1-phosphate as products of the reaction. The forward reaction, conversion of thymidine to thymine, is favored under physiological conditions and it is important in the catabolism of the nucleoside (Fig. 1). Thymidine can be either catabolized by TP to thymine, or phosphorylated by thymidine kinase to thymidine monophosphate. In MNGIE patients, the immediate consequences of TP mutations on the function of the enzyme and the metabolism of thymidine are clear (Table 1). TP activity is not present or it is barely detectable in buffy coats of patients, in contrast to controls that show activities ranging from 400 to

Table 1 Alterations of thymidine metabolism in MNGIE a

MNGIE Heterozygotes Controls a b c d e f

TP activity b (nmol/h/mg prot)

Thd concentration c (mM)

Ratio of urinary elimination of Thd d

Thd in fibroblasts culture medium

2 ^ 5 (N ¼ 27) 236 ^ 75 (N ¼ 29) 667 ^ 205 (N ¼ 19)

8.68 ^ 5.23 (N ¼ 16) ,0.05 (N ¼ 14) ,0.05 (N ¼ 23)

20% (N=2) ND e

Increasing (N ¼ 3) Decreasing (N ¼ 2) Decreasing (N ¼ 3)

TP, thymidine phosphorylase; Thd, thymidine. In buffy coats. In plasma. Referred to urinary elimination of creatinine. ND, not determined. Undetectable thymidine in urine of controls.

f

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1100 nmol/h/mg of protein. The most direct consequence of lack of TP activity in MNGIE patients is the accumulation of its substrate, thymidine, which reaches concentrations between 3 and 25 mM in plasma. In contrast, thymidine is undetectable (,0.05 mM) in both controls and asymptomatic carriers of heterozygous TP mutations (Spinazzola et al., 2002). In healthy individuals, circulating thymidine is undetectable, indicating that cells do not release the nucleoside into the vascular space or alternatively, if some thymidine is released by tissues that do not express TP, the nucleoside is most probably degraded by the enzyme present in platelets, white blood cells and endothelial cells (Fox et al., 1995; Yoshimura et al., 1990). Our experimental findings confirm that in MNGIE patients, released thymidine from tissues contributes to the increased concentrations of this compound in blood. In vitro, fibroblasts of MNGIE patients release this nucleoside into the medium, while control fibroblasts catabolize the thymidine that is initially present, decreasing its concentration (Spinazzola et al., 2002). What is the mechanism that links the disturbances in the thymidine metabolism, which is the direct consequence of absence of TP activity in cells, and the mitochondrial dysfunction observed in this disease? Skeletal muscle does not express TP (Fox et al., 1995; Matsukawa et al., 1996; Yoshimura et al., 1990), and, paradoxically, shows mitochondrial dysfunction. Therefore, the pathomechanism is not restricted to alterations within cells. We hypothesize that high concentrations of thymidine in the blood interfere with normal metabolism of nucleotides, affecting replication of mtDNA.

4. Hypothesis of pathomechanism Mitochondria require dNTP to replicate mtDNA. Even in postmitotic cells, there is mitochondrial division. The organelle has separate and independently regulated dNTP (deoxyribonucleoside triphosphate) pools (Berk and Clayton, 1973; Bestwick and Mathews, 1982; Bestwick et al., 1982), as evident from experimental data demonstrating that mtDNA replication is resistant to agents that block de novo thymidine synthesis compared to nDNA replication (Bogenha-

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gen and Clayton, 1976). According to these observations, mitochondria depend more on the thymidine salvage pathway than on the de novo synthetic pathway. This dependence on the salvage pathway could explain why mitochondrial dNTP levels are more affected by increased concentration of thymidine than the corresponding cytosolic and nuclear pools. Moreover, the existence of a mitochondrial thymidine kinase (TK2), distinct from the cytosolic TK1, probably contribute to the alterations of the mitochondrial dNTP pools (Arne´ r and Eriksson, 1995; Berk and Clayton, 1973; Bestwick et al., 1982; Johansson and Karlsson, 1997). TK1 is upregulated during cell division, and barely expressed or absent in the postmitotic tissues (Arne´ r and Eriksson, 1995); by contrast, TK2 is expressed constitutively. The continuous activity of TK2 could contribute to the susceptibility of mitochondrial dNTPs to the increased levels of thymidine particularly in postmitotic tissue such as muscle. Other evidence points to the same theory. Treatment with nucleoside analogues to patients with viral infections can impair mtDNA replication and produce mtDNA depletion (Arnaudo et al., 1991; Dalakas et al., 1990; Lewis and Dalakas, 1995). A clinical trial for treatment of chronic hepatitis B virus infection with the nucleoside analogue fialuridine, was interrupted, because it produced severe toxicity with lactic acidosis, hepatic failure, hepatic steatosis, skeletal and cardiac myopathy, peripheral neuropathy, and pancreatitis due to inhibition of mtDNA replication in patients (Brahams, 1994; Cui et al., 1995). Recently, mutations in the nuclear genes TK2 and deoxyguanosine kinase (dGK), enzymes involved in mitochondrial dNTP metabolism, have been demonstrated to be cause of two mtDNA depletion syndromes (Mandel et al., 2001; Saada et al., 2001). We hypothesize that mitochondrial dNTP pools are altered leading to mtDNA depletion and multiple deletions in patients with MNGIE. According to this hypothesis, the toxicity of the released thymidine, which cannot be enzymatically eliminated, affects the cells even though they do not express TP. Elimination of circulating thymidine is a direct approach to treat MNGIE patients. Thymidine is filtratable, as is demonstrated by the fact that hemodialysis partially decreases the concentration of the nucleoside in blood (Spinazzola et al., 2002). Thymidine is undetectable in human urine from controls; this result is

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consistent with the fact that thymidine levels in blood are undetectable. MNGIE patients eliminate part of the excess circulating thymidine through urine. Most of the ultrafiltrated thymidine, however, is reabsorbed by the kidney, most likely in the proximal tubule (Gutierrez et al., 1992), and renal clearances were only 20% of creatinine clearance in two patients (Spinazzola et al., 2002). Although hemodialysis effectively reduces thymidine in blood during the treatment, 3 h after the completion of dialysis, thymidine in blood returns to the predialysis levels. Blocking the reabsortion of thymidine by the renal tubules might be more effective than elimination through dialysis.

5. Nucleotides metabolism and mitochondrial diseases MNGIE is the first autosomal disorder, among a group of mitochondrial diseases, to be attributed to disturbances in the nucleoside and nucleotide metabolism. Fig. 2 displays a schematic summary of the known enzymes and transporters that regulate the mitochondrial metabolism of the deoxynucleotides. Some of these proteins have already been reported to be directly involved in maintaining mtDNA. As previously mentioned, two mtDNA depletion syndromes are caused by mutations in TK2 and dGK (Mandel et al., 2001; Saada et al., 2001). Mutations in the adenine nucleotide translocator 1 (ANT1) gene encoding the heart and skeletal muscle isoform of the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) translocator located in the inner mitochondrial membrane, cause autosomal dominant

progressive external ophthalmoplegia (adPEO) and has been hypothesized to possibly cause alterations of dNTP pools (Kaukonen et al., 2000). Other genes related to dNTP have been also indirectly related to mitochondrial dysfunction in human diseases or animal models (Arpaia et al., 2000; Rampazzo et al., 2000). All of these disorders comprise one important part of the group of diseases caused by defects in the cellular intergenomic communication between nDNA and mtDNA (Hirano and Vu, 2000). MNGIE is perhaps a special case because it is the only disease in which mitochondrial nucleoside/nucleotide imbalance is due to mutations in one gene that encodes for a cytosolic protein. To date, the other described disorders involve nDNA encoded mitochondrial proteins (Kaukonen et al., 2000; Mandel et al., 2001; Saada et al., 2001). 6. Conclusion Together with other diseases in which mitochondrial dNTP homeostasis is affected, MNGIE is one among the expanding group of disorders caused by defects in the crosstalk between mitochondrial and nuclear genomes. The fact that these syndromes with alterations of dNTP homeostasis share clinical features and pathophysiological findings provides additional evidence that this is an important subgroup of mitochondrial diseases. The study of each of these diseases will promote our understanding of the molecular mechanisms involved in all, and may lead to novel and effective treatment approaches. References

Fig. 2.

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