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April 16, 2019; 92 (16) Article

Increase of arginine dimethylation correlates with the progression and prognosis of ALS

Kensuke Ikenaka, Naoki Atsuta, Yasuhiro Maeda, Yuji Hotta, Ryoichi Nakamura, Kaori Kawai, Daichi Yokoi, Akihiro Hirakawa, Akira Taniguchi, Mitsuya Morita, Kouichi Mizoguchi, Hideki Mochizuki, Kazunori Kimura, Masahisa Katsuno, Gen Sobue
First published March 13, 2019, DOI: https://doi.org/10.1212/WNL.0000000000007311
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Abstract

Objective To investigate whether arginine methylation is altered in patients with amyotrophic lateral sclerosis (ALS) and how it affects disease severity, progression, and prognosis.

Methods We compared the immunoreactivity of protein arginine methyltransferase 1 (PRMT1) and its products, asymmetric dimethylated proteins (ASYM), in postmortem spinal cord. We also measured the concentrations of total l-arginine and methylated arginine residues, including asymmetric dimethyl l-arginine (ADMA), symmetric dimethyl arginine, and monomethyl arginine, in CSF samples from 52 patients with ALS using liquid chromatography-tandem mass spectrometry, and we examined their relationship with the progression and prognosis of ALS.

Results The immunoreactivity of both PRMT1 (p < 0.0001) and ASYM (p = 0.005) was increased in patients with ALS. The concentration of ADMA in CSF was substantially higher in patients with ALS than in disease controls. The ADMA/l-arginine ratio was correlated with the change of decline in the ALS Functional Rating Scale at 12 months after the time of measurement (r = 0.406, p = 0.010). A Cox proportional hazards model showed that the ADMA/l-arginine ratio was an independent predictor for overall survival. Moreover, a high ADMA/l-arginine ratio predicted poor prognosis, even in a group with normal percentage forced vital capacity.

Conclusion There was an enhancement of arginine dimethylation in patients with ALS, and the ADMA/l-arginine ratio predicted disease progression and prognosis in such patients.

Glossary

ADMA=
asymmetric dimethyl l-arginine;
ALS=
amyotrophic lateral sclerosis;
ALSFRS-R=
ALS Functional Rating Scale;
ASYM=
asymmetric dimethylated proteins;
CI=
confidence interval;
CK=
creatinine kinase;
HR=
hazard ratio;
MMT=
Manual Muscle Testing;
NMMA=
NG-monomethyl l-arginine;
%FVC=
percentage forced vital capacity;
PRMT=
protein arginine methyltransferase;
SDMA=
symmetric dimethyl l-arginine;
TPPV=
tracheostomy positive pressure ventilation

Arginine methylation is a common posttranslational modification of RNA-binding proteins that functions as an epigenetic regulator of transcription and plays key roles in pre-mRNA splicing, mRNA translation, and cell fate decision. Arginine methylation is accomplished by a family of enzymes called protein arginine methyltransferases (PRMTs).1 PRMT1 is the primary methyltransferase and accounts for >90% of asymmetric dimethyl l-arginine (ADMA) production. The major substrates of PRMT1 are RNA-binding proteins such as hnRNPs, FET proteins (FUS, EWS, and TAT15), and ELAV/Hu proteins,2 many of which have been shown to be involved in the pathology of motor neuron diseases.3,4 For example, arginine methylation by PRMT1 has been reported to regulate the cellular localization of fused in sarcoma protein (FUS), stress granule formation, and cellular toxicity of amyotrophic lateral sclerosis (ALS)-linked FUS mutants.5,–,7 We previously reported that PRMT1 is expressed mainly in neurons during the early embryonic stage and that its expression is suppressed in the adult CNS8; however, PRMT1 expression can be induced again in response to various types of stress, including oxidative stress, hypoxia, and mitochondrial stress.9,–,14

In this study, we investigated whether patients with ALS have altered arginine methylation by PRMT1 and evaluated the prognostic significance of dimethylated arginine residues in CSF as a candidate pathologic marker for ALS.

Methods

Immunohistochemistry

We performed immunohistochemical analysis as described previously.15,16 In brief, 6-µm-thick sections were prepared from paraffin-embedded spinal cord sections from autopsied patients.16 Four patients with sporadic ALS (age 64.5 ± 9.3 years, male:female 2:2) and 4 disease controls (age 73.5 ± 5.4 years, male:female 1:3), who were not statistically different, were included in the immunohistochemical analysis. The 4 control patients had diagnoses of progressive supranuclear palsy, multiple system atrophy, diffuse Lewy body disease, and Parkinson disease. The postmortem interval until dissection was not significantly different between both groups (control patients 7.338 ± 2.632 hours, patients with ALS 6.343 ± 1.537 hours). The cause of death for the control patients was sepsis, renal failure, multiple organ failure, and respiratory failure, while all of the patients with ALS died of respiratory failure. The sections were microwaved for 20 minutes in 50 mmol/L citrate buffer, pH 6.0, blocked with TNB blocking buffer (PerkinElmer, Hvidovre, Denmark) in Tris-buffered saline (pH 7.5), and incubated with a monoclonal antibody against PRMT1 (anti-PRMT1, 1:1,000, Abcam, Cambridge, MA) or asymmetric dimethylated proteins (ASYM) (anti-ASYM24, 1:2,000, Millipore, Bedford, MA) overnight at 4°C. We performed the subsequent procedures using an EnVision+ Kit/HRP (DAB) (DAKO, Glostrup, Denmark) according to the manufacturer's protocol. We expressed signal intensity as the individual intracellular signal level (arbitrary absorbance units per 1 mm2) per motor neuron in each slide.

Patient registry and follow-up

We prospectively enrolled patients diagnosed as having ALS at Nagoya University, Shizuoka-Fuji National Hospital, Jichi Medical University, or Mie University from March 2007 to August 2012. In total, 52 patients with ALS with definite (n = 7), probable (n = 25), probable laboratory-supported (n = 16), or possible (n = 4) ALS according to the revised El Escorial criteria were included. We also registered the included patients in the Japanese Consortium for ALS Research, a multicenter ALS patient registry in Japan, and assessed the patients using the follow-up system of the registry. The precise system of the registry and the follow-up system have been described previously.19,20 We tested and scored muscle strength using the Medical Research Council Manual Muscle Testing (MMT) score (6 points, range 0–5).17 We categorized MMT into the 3 following groups: neck flexor muscles as representative of the MMT neck score; bilateral abductor muscles of the shoulders, elbow flexor muscles, and wrist extensor muscles as representative of the MMT upper limb score; and bilateral flexor muscles of the hip and ankle dorsiflexion muscles as representative of the MMT lower limb score.

We defined disease onset as the time when the patients became initially aware of muscle weakness or impairment of swallowing, speech, or respiration. We used the Japanese version of the ALS Functional Rating Scale (ALSFRS-R)21 as a scale for the activities of daily living. The reliability of the follow-up system for the Japanese version of the ALSFRS-R has been confirmed previously.22 To evaluate the functional decline of clinical scores such as ALSFRS-R and percentage forced vital capacity (%FVC), we calculated the slope defined as decline of the value within a specific duration divided by duration. For example, we defined the 12-month ALSFRS-R slope as decline of the ALSFRS-R score at 12 months after diagnosis divided by 12. When a patient died within 12 months, we used the ALSFRS-R slope obtained at the last point before death. We also used the preslope to evaluate the decline of the ALSFRS-R score from onset to diagnosis (registration), which was calculated as follows: ALSFRS-R preslope = (48 − ALSFRS-R at registration)/(duration from onset to diagnosis). We also evaluated the decline of %FVC from onset to diagnosis (registration) as follows: %FVC preslope = (100 − %FVC at registration)/(duration from onset to diagnosis). If %FVC > 100%, the preslope is taken as zero.

We defined the introduction of tracheostomy positive pressure ventilation (TPPV) or death of the patient as the primary endpoint, and we determined the primary endpoint by the follow-up system. We defined TPPV-free survival as survival in the TPPV cases.

Clinical data of patients with ALS and controls

The average age at registration, sex ratio, and total protein concentration in CSF were not significantly different between patients with ALS and controls (age: ALS 65.0 [58.5–71.5] years and controls 62.0 [48.5–71.5] years; sex [male:female]: ALS 32:20 and controls 12:8; and CSF protein: ALS 49.8 [35.0–69.0] mg/dL and controls 44.7 [35.0–57.0] mg/dL). The average duration from onset to registration in patients with ALS was 13.6 (10.7–25.0) months; the average ALSFRS-R score at registration was 39.0 (34.5–43.0); and the %FVC at registration was 92.5 (73.2–105.2), which are consistent with previous studies.18,–,21 At the end of this study, among the 52 participants, 28 patients reached the primary endpoint, and the average duration from registration to the primary endpoint was 17.13 months. For disease form (initial symptoms), 10 patients showed the bulbar form and 42 patients showed the spinal form. As a disease control, we used 21 patients with Parkinson disease (n = 6), multiple system atrophy (n = 3), chronic inflammatory demyelinating polyneuropathy (n = 3), myositis (n = 2), dystonia (n = 1), multiple sclerosis (n = 1), oculomotor palsy (n = 1), benign muscle atrophy (n = 1), spinal sarcoidosis (n = 1), minor cognitive impairment (n = 1), and anhidrosis (n = 1).

Measurement of arginine analogs

We used arginine, NG, NG-dimethyl l-arginine (ADMA), NG, N′G-dimethyl l-arginine (symmetric dimethyl l-arginine [SDMA]), and NG-monomethyl l-arginine (NMMA) to construct standard curves (Enzo Life Sciences, Farmingdale, NY). We purchased 13C6, 15N4-arginine, which we used as an internal standard, from Sigma-Aldrich (St. Louis, MO). We prepared ADMA-d6, SDMA-d6, and NMMA-d3 according to a previously described method.22

We measured these arginine analogs using a high-performance liquid chromatography-tandem mass spectrometry system (Quattro Premier XE Mass Spectrometer; Waters Corp, Milford, MA). We injected a 5-μL sample solution that was deproteinated by methanol into an Intrada Amino Acid Column (2 × 50 mm; Imtakt, Kyoto, Japan) at 40°C. We performed chromatography at a flow rate of 0.6 mL/min using a step gradient alternating between a mixture of acetonitrile:tetrahydrofuran:25 mmol/L aqueous ammonium formate:formic acid (9:25:16:0.3) and a mixture of 100 mmol/L aqueous ammonium formate:acetonitrile (80:20). We analyzed arginine analogs using the multiple reaction monitoring mode of tandem mass spectrometry in positive ion mode. The cone voltage was 22 to 25 V; collision energy was 13 to 22; and transitions were m/z 175 → 70 for arginine, m/z 203 → 46 for ADMA, m/z 203 → 172 for SDMA, and m/z 189 → 70 for NMMA.

Statistical analysis

We used a 2-sample t test to compare continuous variables between 2 groups. We defined survival time as the time from disease onset to death or the introduction of TPPV. We used the Kaplan-Meier method to estimate survival curves, and we compared the survival curves of 2 groups by using the log-rank test. We applied a Cox proportional hazard model, which included age at registration (years), sex (male vs female), duration from onset to registration (months), ALSFRS-R score at registration, MMT score at registration, and %FVC at registration, to evaluate the effect of these variables on survival time. We estimated the hazard ratio (HR) and 95% confidence interval (CI). We also performed multivariate regression analyses with stepwise variable selection (α = 0.05 for inclusion and α = 0.10 for exclusion) to evaluate the effect of the ADMA/l-arginine ratio on functional decline. We used analysis of covariance in tables 1 through 4 to adjust the data for a covariate. We also used 1-way analysis of variance with the Bonferroni post hoc test for multiple-comparison tests. We considered values of p < 0.05 significant and correlation coefficients (r) >0.4 as sufficient. We used Statistical Package for the Social Sciences 23.0J software (IBM Japan, Tokyo, Japan) to perform statistical analysis.

View this table:
Table 1

Multivariate Cox regression analysis with adjustments of the covariates (ADMA/l-arginine ratio vs clinical scores)

View this table:
Table 2

Multivariate Cox regression analysis with adjustments of the covariates (serum biological markers)

View this table:
Table 3

Multivariate linear regression analysis with stepwise variable selection

View this table:
Table 4

Multivariate linear regression analysis with stepwise variable selection (serum biological markers)

Bias

Our data are from patients who agreed to participate in the Japanese Consortium for ALS Research study and agreed to receive a lumbar puncture for the diagnosis of ALS.

Standard protocol approvals, registrations, and patient consents

We conducted this study in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects endorsed by the Japanese government. We registered the patients in the participating facilities with written informed consent. The ethics committees of all participating institutions approved the study. The Ethics Committee of Nagoya University Graduate School of Medicine approved the collection of autopsied human tissues and their use in this study.

Data availability

We deposited all raw data used in this study in the UMIN Individual Case Data Repository, and all data are publicly available to other researchers. Further details of our data sharing policy are available at umin.ac.jp/icdr/index.html.

Results

Dysregulation of protein arginine dimethylation in postmortem spinal cord tissues from patients with ALS

We first examined the expression of PRMT1 and its substrates containing ASYM in the spinal cord of patients with ALS by immunohistochemistry. The immunoreactivity of both PRMT1 and proteins containing dimethylated arginine residues increased in the spinal cord of patients with ALS (figure 1A). Next, we performed immunofluorescent double staining with PRMT1 and choline acetyltransferase and with ASYM and choline acetyltransferase using consecutive spinal cord sections, which revealed that PRMT1 (p < 0.0001) and ASYM (p = 0.005) were increased in the spinal motor neurons of patients with ALS (figure 1, B–D).

Figure 1
Figure 1 Dysregulated expression of PRMT1 and ASYM in the spinal cord of patients with ALS

(A and B) Representative (A) immunohistochemistry and (B) immunofluorescence analyses of protein arginine methyltransferase 1 (PRMT1) and asymmetric dimethylated proteins (ASYM) from controls and patients with amyotrophic lateral sclerosis (ALS). (C and D) Quantification of the signal intensity of PRMT1 and ASYM in choline acetyltransferase–positive neurons in the anterior horn of the spinal cord (n = 20 sections from 4 patients for each group). Scale bar = 50 μm (A and B). We performed statistical analysis using the Student t test (**p < 0.01 and ***p < 0.001).

Increased ADMA and l-arginine concentration in the CSF of patients with ALS

After proteolytic degradation of methylated intracellular proteins, free monomethyl l-arginine, SDMA, or ADMA can be released from cells into the extracellular space such as CSF.23 We analyzed the CSF concentration of these methylated arginine and nonmethylated l-arginine residues (figure 2, A–D). In CSF, the concentrations of ADMA and nonmethylated l-arginine increased significantly in patients with ALS (figure 2, A and D).

Figure 2
Figure 2 Increased ADMA/l-arginine ratio in CSF from patients with ALS and its relationship with disease prognosis

(A–D) Asymmetric dimethyl l-arginine (ADMA), symmetric dimethyl l-arginine (SDMA), monomethyl l-arginine (MMA), and l-arginine CSF concentrations of controls and patients with amyotrophic lateral sclerosis (ALS). Patients with ALS had higher CSF ADMA and l-arginine concentrations. We performed statistical analysis using the Student t test. (E) ADMA/l-arginine ratio in the CSF of controls and patients with ALS. (F) Correlation between CSF ADMA and l-arginine. Red circles indicate patients with ALS; blue circles indicate control patients. Red line is the regression line for patients with ALS. (G) Kaplan-Meier curve according to the ADMA/l-arginine ratio (low: <5.0412, blue line; high: >5.0412, red line). We compared the Kaplan-Meier curves for the primary endpoint by the log-rank test. Difference of the curves was significant (p < 0.0001). (H) Correlation of the ADMA/l-arginine ratio with the duration until primary endpoint.

The ADMA/l-arginine ratio is an independent predictor for survival

Because l-arginine antagonizes the toxic effect of ADMA, and the ratio of ADMA and l-arginine is biologically significant,24 we analyzed the ADMA/l-arginine ratio in patients with ALS (figure 2, E and F). Although the average ADMA/l-arginine ratio was not significantly different between patients with ALS and disease controls, correlation analysis between ADMA and l-arginine revealed that the ALS group had a wider distribution of ADMA and l-arginine than the control group, indicating that the ADMA/l-arginine ratio could be a useful measure of disease severity or progression.

Indeed, multivariate Cox regression analysis of survival time (table 1) revealed an association between the ADMA/l-arginine ratio and poor survival. Conversely, ADMA or l-arginine alone was not associated with poor survival (HR 1.012, 95% CI 0.954–1.073, p = 0.694 for ADMA; HR 1.000, 95% CI 0.999–1.000, p = 0.209 for l-arginine). Coanalysis of the ADMA/l-arginine ratio with serum markers such as creatinine kinase (CK), creatinine, and albumin also showed that the ADMA/l-arginine ratio and serum creatinine were independent predictors of patient survival (table 2).

We then explored the optimal cutoff score for the ADMA/l-arginine ratio to predict the prognosis of patients with ALS. When we used a cutoff of the ADMA/l-arginine ratio >5.0412, the HR was 5.849 (95% CI 1.804–18.965, p < 0.003). We divided the registered patients into 2 categories using this cutoff score (ADMA/l-arginine 5.0412). Figure 2G shows the Kaplan-Meier curves for the 2 categories for the primary endpoint. The curves were statistically different according to a log-rank test (p < 0.0001). We also analyzed the subgroup of patients who reached the primary endpoint during our investigation (n = 28). The duration until primary endpoint was shorter in the patients with higher ADMA/l-arginine ratio (r = −0.512, p = 0.005) (figure 2H).

The ADMA/l-arginine ratio is an independent predictor for the functional decline of patients with ALS

We analyzed whether the ADMA/l-arginine ratio predicted functional decline, as measured by the ALSFRS-R slope (see Methods). Multivariate linear regression analysis revealed that among the ADMA/l-arginine ratio, clinical scores described above, or serum markers, only the ADMA/l-arginine ratio independently predicted the decline of the ALSFRS-R slope (tables 3 and 4).

Although the ADMA/l-arginine ratio was sufficiently correlated with the ALSFRS-R slope (figure 3A) and ALSFRS-R preslope (figure 3B), it was not associated with the ALSFRS-R score at registration (figure 3C) or with the total MMT score at registration (figure 3D). This result showed that the ADMA/l-arginine ratio had a closer relationship to disease progression than to the functional score at the point of examination. We confirmed that serum creatinine, which is associated with muscle volume,25 showed a strong correlation with the ALSFRS-R score at registration (r = 0.434, p = 0.027) and the MMT total score at registration (r = 0.523, p = 0.006), but not with the ALS progression scores such as the 12-month ALSFRS-R slope (r = −0.279, p = 0.151) or ALSFRS-R preslope (r = 0.141, p = 0.491).

Figure 3
Figure 3 ADMA/l-arginine ratio predicts the functional decline of patients with ALS

(A–C) Correlation of the (A–C) asymmetric dimethyl l-arginine (ADMA)/l-arginine ratio with (A and B) disease progression scores (ALS Functional Rating Scale [ALSFRS-R] slope and preslope), (C) ALSFRS-R, (D) Manual Muscle Testing (MMT) total, (E) percentage forced vital capacity (%FVC), or (F) %FVC preslope in patients with amyotrophic lateral sclerosis (ALS). (G) ADMA/l-arginine ratio was compared among the subgroups of MMT neck score. ADMA/l-arginine ratio was higher in patients with a lower MMT neck flexion score. We performed statistical analysis by 1-way analysis of variance followed by the Bonferroni post hoc comparisons test (p = 0.002 and p = 0.005, MMT neck score 2 vs 4 and 2 vs 5, respectively). (H–K) Kaplan-Meier curve in patients with ALS with %FVC > 80%. The patients with %FVC > 80% were divided (H) by the median ADMA/l-arginine ratio (n = 28, median 4.48), (I) by the median %FVC (n = 28, median = 103.1), (J) by the %FVC preslope (n = 28, divided by %FVC preslope 0 or >0), or (K) by the median ALSFRS-R preslope (n = 28, median 0.451). We compared the Kaplan-Meier curves for the primary endpoint by the log-rank test. Only the curve divided by the ADMA/l-arginine ratio was statistically different (p = 0.046).

We also analyzed the correlations of several clinical scores (age, MMT total score, %FVC, ALSFRS-R, and disease duration until registration) and serum biological markers (CK, creatinine, and albumin) with the ADMA/l-arginine ratio. The ADMA/l-arginine ratio was correlated only with %FVC (r = −0.464, p = 0.001) (figure 3E). We also found that the decline of %FVC (%FVC preslope, see Methods) was faster in patients with a higher ADMA/l-arginine ratio (figure 3F). Therefore, we hypothesized that the increase of the ADMA/l-arginine ratio had a relationship with respiratory failure.

Consistent with this idea, multivariate linear regression analysis revealed that only a decrease of the MMT neck flexion score contributed to an increase of the ADMA/l-arginine ratio among the MMT scores (neck, upper limbs, and lower limbs) (MMT neck: coefficient −0.815 [95% CI −1.312 to −0.318], p = 0.002). In addition, 1-way analysis of variance followed by the Bonferroni post hoc test revealed that the ADMA/l-arginine ratio was higher in patients whose MMT neck score was 2 (figure 3G). Because the respiratory muscles and neck flexion muscles are both similarly innervated by higher cervical cord regions, it makes sense that the MMT neck flexion score was also associated with the ADMA/l-arginine ratio.20

The ADMA/l-arginine ratio predicts the prognosis for early-stage patients with normal %FVC

Because the ADMA/l-arginine ratio was well correlated with %FVC and %FVC is a strong predictor for survival,26,–,28 we examined whether the ADMA/l-arginine ratio can predict the prognosis of patients with normal %FVC (>80%). We divided the patients according to the median ADMA/l-arginine ratio (n = 14, and 14, average ADMA/l-arginine ratio 6.14 ± 2.53 and 3.43 ± 0.71, respectively) and analyzed the survival curve of each group. As shown in figure 3H, even in the normal %FVC group, patients with a higher ADMA/l-arginine ratio showed a poorer prognosis (log-rank test, p = 0.046). Conversely, when we divided the patients with normal %FVC by median %FVC (n = 14, 14, average %FVC 94.4 ± 7.89% and 103.9 ± 8.12%, respectively), there was no difference in the survival curves of both groups (log-rank test, p = 0.844) (figure 3I). These results suggest that the ADMA/l-arginine ratio is a more sensitive marker for survival than %FVC.

Discussion

In this study, we investigated the involvement of altered arginine dimethylation in patients with ALS. In a prospective multicenter study of CSF, we found that the ADMA/l-arginine ratio is a potent predictor of survival and functional decline in patients with ALS. We showed that even in a group with normal %FVC, a higher ADMA/l-arginine ratio predicted a poorer prognosis, indicating that the ADMA/l-arginine ratio is a highly sensitive biomarker for the early stage of the disease.

Why did the ADMA/l-arginine ratio not correlate with the ALSFRS-R score, MMT score, serum creatinine, or serum CK, and why was it strongly correlated only with %FVC? We think that total muscle volume affects serum creatinine and CK levels, and the ALSFRS-R or total MMT scores are more likely to reflect systemic motor function, indicating total muscle volume. Conversely, the correlation between the CSF ADMA/l-arginine ratio and %FVC or the MMT neck flexion score indicated the possibility that respiratory failure and hypoxia in the CNS have a role in the increase of the ADMA/l-arginine ratio in CSF.

At the same time, the ADMA/l-arginine ratio was a more sensitive marker than %FVC or %FVC preslope, indicating the possibility that it not only reflects respiratory failure but also affects the progression of disease through other mechanisms such as affecting the RNA-binding protein function or aggregation. Further studies are required to show directly the relationship between ADMA/l-arginine dysregulation and progression of ALS.

Our results showed the importance of the ADMA/l-arginine ratio. This result is consistent with previous studies showing that the ADMA/l-arginine ratio is a potential predictor of the prognosis of several systemic diseases such as cardiovascular diseases,29 atherosclerotic plaques,30 and chronic renal failure.31 The ratio of ADMA and l-arginine is important because increased ADMA inhibits the activity of nitric oxide synthase and compensates with nitric oxide production from l-arginine. In support of this idea, recent studies showed that numerous signs of blood-CNS-barrier impairment, for example, endothelial cell degeneration, capillary leakage, perivascular edema, downregulation of tight junction proteins, and microhemorrhages, are observed in a mutant SOD1 ALS model32 and in patients with sporadic ALS.33,34 Although we still need to identify the pathomechanisms by which the increased ADMA/l-arginine ratio affects disease progression, it is possible that dysregulation of the ADMA/l-arginine ratio can be a novel therapeutic target for ALS.

Some limitations of the present study merit discussion. In immunohistochemical analysis of postmortem tissues, we assessed a small number of samples; we did not examine the effect of respiratory failure before death, which is prevalent in patients with ALS; and the stability of PRMT1 and methylated arginine in the period after death until fixation is unknown. For the CSF study, we need to validate our findings analytically and in the clinical setting to determine the generalizability of this biomarker.

Study funding

This work was supported by grants (15Aek0109071h0002 and 17ek0109284h0001) from the Japan Agency for Medical Research and Development, and the Health and Labor Sciences Research grant (H26-086), and Grants-in Aid (25461277 and 17K09778) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. A section of this study is the result of the Integrated Research on Neuropsychiatric Disorders study, which was performed under the Strategic Research Program for Brain Sciences by MEXT, Japan.

Disclosure

The authors report no disclosures relevant to the manuscript. Go to Neurology.org/N for full disclosures.

Appendix Authors

Table
Table

Footnotes

  • Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

  • Received July 5, 2018.
  • Accepted in final form December 17, 2018.

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