Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS)

Journal Pre-proofs Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS) Takamasa Nukui, Atsushi Matsui, Hideki Niimi, Mamoru Yamamoto, Noriyuki Mastuda, Jin-Lan Piao, Kyo Noguchi, Isao Kitajima, Yuji Nakastuji PII: DOI: Reference:

S1567-7249(19)30216-8 https://doi.org/10.1016/j.mito.2019.11.001 MITOCH 1424

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Mitochondrion

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15 August 2019 1 November 2019

Please cite this article as: Nukui, T., Matsui, A., Niimi, H., Yamamoto, M., Mastuda, N., Piao, J-L., Noguchi, K., Kitajima, I., Nakastuji, Y., Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS), Mitochondrion (2019), doi: https:// doi.org/10.1016/j.mito.2019.11.001

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Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS)

Takamasa Nukui a, Atsushi Matsui b, Hideki Niimi c, Mamoru Yamamoto a, Noriyuki Mastuda a, JinLan Piao a, Kyo Noguchi d, Isao Kitajima c, Yuji Nakastuji a*

a Department b First

of Neurology, Toyama University hospital

Department of Internal Medicine, Toyama University Hospital

c Department

of Clinical Laboratory and Molecular Pathology, Graduate School of Medicine and

Pharmaceutical Science for Research, University of Toyama d Department

of Radiology, Graduate School of Medicine and Pharmaceutical

Science, University of Toyama

*Author for correspondence: Yuji Nakatsuji Department of Neurology, Toyama University Hospital. 2630 Sugitani, Toyama, 930-0194, Japan Phone: +81-76-434-7309, Fax +81-76-434-5033 e-mail: [email protected]

1

Abstract Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by defective oxidative phosphorylation in the cerebral parenchyma, cerebral blood vessels, and leptomeningeal tissue. Although increased serum and cerebrospinal fluid (CSF) lactate level has been used as a diagnostic biomarker in patients with MELAS, no biomarkers reflecting disease activity exist. Since we have developed a highly sensitive ATP assay system using luciferase luminous reaction, we examined CSF ATP in patients with MELAS and found that it negatively correlates with disease activity and that it reflects the efficacy of the treatment. CSF ATP might be a novel disease monitoring marker for MELAS.

Key words:

MELAS, biomarker, ATP, cerebrospinal fluid

Introduction Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by impaired mitochondrial energy production (Craven et al. 2017). Serum and cerebrospinal fluid (CSF) lactate levels are increased in patients with MELAS, reflecting defective oxidative phosphorylation in mitochondria (Debray et al. 2007). Serum and CSF lactate level has therefore been used as a diagnostic biomarker for MELAS (Mager et al. 2011). In addition, it is reported to correlate with the severity of the disease (Robinson et al. 2006; Kaufmann et al. 2011). However, lactate levels are not always upregulated in patients with MELAS; therefore, other biomarkers such as fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) have been investigated as biomarkers for diagnosis and disease activity monitoring (Suomalainen et al. 2011; Yatsuga et al. 2015). As the impaired mitochondria of brain tissues cannot generate sufficient adenosine triphosphate (ATP) in MELAS patients (King et al. 1992; Weiduschat N et al. 2014), it is reasonable to assume that CSF ATP would decrease in accordance with the disease severity. We therefore 2

assayed the levels of CSF ATP chronologically with a newly established, highly sensitive assay system using luciferase luminous reaction. .

Materials and Methods Patients and ethics Two patients with MELAS admitted to Toyama University hospital were enrolled in this study. Blood and CSF tests, including CSF lactate and ATP levels, and brain MRI scans were performed to monitor the disease progression. In addition, 46 patients with other neurological diseases including 25 multiple sclerosis (MS), 16 amyotrophic lateral sclerosis (ALS), and 5 idiopathic normal pressure hydrocephalus (iNPH) patients were enrolled as control. The present study was performed with the approval of the Ethics Committee of the University of Toyama (approval no. 29-32). Written informed consent was obtained from patients. Patient 1 was a 68-year-old woman exhibiting consciousness disturbance and sensory aphasia. She had experienced several similar exacerbations of consciousness disturbance and sensory aphasia in previous several years. On admission, brain MRI showed high intensity lesions in left temporal, parietal, and occipital lobes of the cortex on diffusionweighted image, and low intensity lesions on an apparent diffusion coefficient (ADC) map (Fig. 1 A, B). These lesions represented high intensity on T2/FLAIR images and gadolinium-enhanced T1 images (Fig. 1C, D). Although the serum lactate level of 14.1 mg/dL was within the normal range, the CSF lactate level of 27.5 mg/dL was elevated. Magnetic resonance spectroscopy (MRS) showed an elevated lactate peak in the occipital lesion (Fig. 1E). Genetic sequencing analysis demonstrated an A>G point mutation at position 3243 of the mtDNA in the whole blood sample. Based on these findings and the criteria described in Yatsuga et al. (2012), patient 1 was diagnosed with MELAS. Intravenous edaravone administration decreased the severity of consciousness disturbance, followed by a gradual improvement of sensory aphasia symptoms by the administration of L-arginine, 3

ubidecarenone, and second cycle of edaravone (Fig. 2). At the time of discharge, gadolinium enhancement of left temporal, parietal, and occipital lobe cortices had disappeared and T2/FLAIR high intensity lesions were detected in both cortices and white matter (Fig. 1F, G). Patient 2 was a 68-year-old woman exhibiting nausea and myoclonic seizure of the right upper limb. Brain MRI revealed multiple high intensity lesions in the left temporal, right frontal, and left parietal lobe cortices on T2/FLAIR images (Fig. 3A - C). Serum lactate was slightly elevated at 17.1 mg/dL at rest and increased to 24.6 mg/dL following a ten-minute aerobic exercise test. MRS showed an elevated lactate peak in the right frontal lobe lesion (Fig. 3D). Whereas genetic analysis of whole blood sample did not show an A>G point mutation at position 3243, a right brachial bicep muscle biopsy revealed the cytochrome c oxidase deficient ragged-red fibers, which in conjunction with encephalopathy is indicative of MELAS. Patient 2 was thus diagnosed with MELAS. Intravenous edaravone administration ameliorated nausea and myoclonic seizure, but upper right quadrantanopia persisted and several new brain lesions appeared. Although a second cycle of edaravone was administered, brain lesions further increased, and MELAS symptoms were exacerbated. Subsequently, L-arginine was administered, which reduced the severity of nausea, myoclonic seizure, and upper right quadrantanopia. Brain lesions had also decreased in number and size at discharge (Fig. 4).

Measurement of the extracellular ATP levels A highly sensitive and automated ATP measurement device was used to measure the extracellular ATP level (Okanojo et al. 2017). We selected the Luciferin-luciferase reagent HS attached to Lucifell HS Set (Kikkoman Biochemifa Co., Ltd., Tokyo, Japan) as a luminescent reagent and diluted it 10-fold with distilled water. The CSF specimens were diluted 10-fold with distilled water prior to measurement in order to prevent the inhibitory activity of high concentrations of chloride ions on the luminescent reagent. Luminescence was measured by the luminometer for 10 seconds after the addition of 50 µL 4

of luminescent reagent to 10 µL of diluted specimen. We determined the extracellular ATP level as the average of relative light intensities (count per second). The measurement was performed three times for each sample. A calibration curve of luminescence and ATP concentration was obtained using a 10-fold dilution series of standard ATP solution adjusted with a saline solution diluted 10-fold with distilled water. The ATP concentration was calculated from the luminescence value obtained from the calibration curve.

Results In patient 1, CSF ATP was 14 pmol/L on admission (day 0). After the first cycle of edaravone administration, CSF ATP increased to 418 pmol/L, which coincided with a decrease in the severity of the consciousness disturbance observed on day 9. Subsequently, CSF ATP slightly increased to 442 pmol/L with the initiation of L-arginine and ubidecarenone administration on day 39. A second cycle of edaravone was administered, and upon discharge on day 59, CSF ATP had increased to 1868 pmol/L with slight remission of sensory aphasia. During the clinical course, CSF lactate levels remained within the high range between 24.0 mg/dL to 27.5 mg/dL (Fig. 2). In patient 2, CSF ATP was 3,205 pmol/L on admission (day 0). Although two cycles of edaravone were administered, CSF ATP decreased to 64 pmol/L on day 48 accompanied by exacerbation of the brain lesions and related symptoms. Subsequent to L-arginine administration, CSF ATP increased to 5,370 pmol/L on day 86, which coincided with a reduction of brain lesion severity and related symptoms. CSF lactate remained within a normal range between 13.3 – 15.4 mg/dL during the clinical course (Fig. 4). Thus, CSF ATP levels seemed to reflect disease activity and the efficacy of the treatment. We also measured CSF ATP concentrations of 46 patients with other neurological diseases including MS, ALS, and iNPH as control. The mean concentration was 3,134±832 (SEM) pmol/L. 5

Discussion We demonstrated that an increase in CSF ATP levels correlates with the clinical amelioration of symptoms in two patients with MELAS. CSF ATP levels were below the normal range on admission and gradually increased in accordance with the several medications. Patient 1 experienced an increase in CSF ATP levels and clinical amelioration of symptoms; whereas patient 2 initially experienced a decrease in CSF ATP and an exacerbation of brain lesions. With subsequent treatment, CSF ATP levels of patient 2 returned to admission levels along with a decrease in the severity of brain lesions and the clinical amelioration of related symptoms. In patients with MELAS, dysfunctional mitochondria cannot generate sufficient ATP to meet the requirements of neural cells, which results in stroke-like episodes (El-Hattab et al. 2015; Goto et al. 1992). It is thus assumed that decreased CSF ATP reflects the failure of ATP production in the brain. To the best of our knowledge, no previous reports have evaluated the CSF ATP in patients with MELAS. Rodan et al. demonstrated that muscle ATP levels detected by 31P-MRS were lower in MELAS patients than in healthy controls (Rodan et al. 2015). It thus seems likely that decreased CSF ATP level reflects reduced ATP synthesis by the central nervous system of patients with MELAS who developed stroke-like episodes. Because patient 1 had experienced repeated stroke-like episodes in the past few years before admission, ATP levels in the brain were chronically reduced, and CSF ATP was at the lowest level on admission. In the case of patient 2, the first stroke-like episode with small brain lesions occurred on admission, and therefore it is assumed that brain mitochondrial function was still preserved and CSF ATP had not yet decreased. Because the mean CSF ATP concentration of 46 patients with other neurological diseases was 3,134±832 (SEM) pmol/L, the levels in our two MELAS patients at discharge (Patient 1; 1868 pmol/L, Patient 2; 5370 pmol/L) were not so different from those in other neurological diseases. CSF lactate levels remained high with no correlation to the symptomatic improvement in patient 1 (Fig. 2). In patient 2, CSF lactate levels remained normal without correlation to the exacerbated 6

clinical symptoms and brain lesions (Fig. 4). Hirano et.al reported that serum and CSF lactate levels can be normal in some MELAS cases (Hirano et al. 1994). In such cases, serum and CSF lactate levels are not useful biomarkers for diagnosing the disease and monitoring disease progression or efficacy of treatment. Interestingly, in this study the levels of CSF ATP inversely correlated with the clinical severity of the patients. Thus, CSF ATP levels might be a more sensitive biomarker than lactate in patients with MELAS. In summary, we demonstrated an inverse correlation between CSF ATP and disease severity that reflected the efficacy of the treatment. This indicates that CSF ATP may be a useful biomarker for monitoring the treatment of patients with MELAS.

Further reports should be accumulated to

determine whether CSF ATP may be a useful biomarker for treating MELAS patients.

Acknowledgments This study is supported in part by the Health and Labour Sciences Research Grant on Rare and Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan and by the Practical Research Project for Rare/Intractable Diseases of the Japan Agency for Medical Research and Development (AMED).

Disclosure of conflicts of interest The authors declare no conflicts of interest.

Figure legends Fig. 1. Brain MRI images upon admission to hospital. On admission, patient 1 showed high intensity lesions in left temporal, parietal, occipital lobe cortices on diffusion-weighted image (A), and low intensity signal on an ADC map (B). These lesions exhibited high intensity on FLAIR images (C), gadolinium-enhanced T1 image (D). MRS showed elevated lactate levels in the 7

occipital lesion (E). Brain MRI at discharge showed FLAIR high intensity lesions in the left temporal, parietal, and occipital lobe cortices and white matter (F). Gadolinium enhancement in these lesions was not visible at discharge (G). The lesions are indicated by arrowheads.

Fig. 2. Therapeutic course, CSF lactate, and ATP levels during treatment period in patient 1.

Fig. 3. Brain MRI showed multiple high intensity lesions in the left temporal, right frontal, and left parietal lobe cortices on FLAIR images (A-C). MRS showed the elevated lactate peak in the right frontal lesion (D). The lesions are indicated by arrowheads.

Fig. 4. Therapeutic course, CSF lactate, and ATP levels during treatment period in patient 2.

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12. Suomalainen A, Elo JM, Pietilainen KH, et al. FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies; a diagnostic study. Lancet neurol 2011; 10(9): 806818. 13. Weiduschat N, Kaufmann P, Mao X, et al. Cerebral metabolic abnormalities in A3243G mitochondrial DNA mutation carriers. Neurology 2014; 82(9): 798-805. 14. Yatsuga S, Fujita Y, Ishii A, et al. Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders. Ann. Neurol 2015; 78(5): 814-823. 15. Yatsuga S, Povalko N, Nishioka J, Katayama K, Kakimoto N, Matsuishi T, et al. MELAS: a nationwide prospective cohort study of 96 patients in Japan. Biochim Biophys Acta. 2012; 1820(5): 619-24. Abstract Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by defective oxidative phosphorylation in the cerebral parenchyma, cerebral blood vessels, and leptomeningeal tissue. Although increased serum and cerebrospinal fluid (CSF) lactate level has been used as a diagnostic biomarker in patients with MELAS, no biomarkers reflecting disease activity exist. Since we have developed a highly sensitive ATP assay system using luciferase luminous reaction, we examined CSF ATP in patients with MELAS and found that it negatively correlates with disease activity and that it reflects the efficacy of the treatment. CSF ATP might be a 9

novel disease monitoring marker for MELAS.

Key words:

MELAS, biomarker, ATP, cerebrospinal fluid

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