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Original Investigation |

Association of Cerebrospinal Fluid Levels of Lateral Olfactory Tract Usher Substance (LOTUS) With Disease Activity in Multiple Sclerosis FREE

Keita Takahashi, MD1,2; Yuji Kurihara, PhD1; Yume Suzuki, MD, PhD2; Yoshio Goshima, MD, PhD3; Fumiaki Tanaka, MD, PhD2; Kohtaro Takei, PhD1
[+] Author Affiliations
1Molecular Medical Bioscience Laboratory, Department of Medical Life Science, Yokohama City University Graduate School of Medical Life Science, Yokohama, Japan
2Department of Neurology and Stroke Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan
3Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
JAMA Neurol. 2015;72(2):176-179. doi:10.1001/jamaneurol.2014.3613.
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Importance  Although multiple sclerosis (MS) is generally considered an autoimmune demyelinating disorder of the central nervous system, axonal degeneration through Nogo receptor-1 signaling was recently recognized as an important pathological feature. Our previous identification of lateral olfactory tract usher substance (LOTUS), an endogenous Nogo receptor-1 antagonist, prompted us to analyze the relationship between LOTUS levels of cerebrospinal fluid and the clinical course of MS to evaluate whether LOTUS could be a useful biomarker for MS.

Objective  To examine variations in LOTUS concentrations in the cerebrospinal fluid of patients with MS in accordance with their clinical course.

Design, Setting, and Participants  Cerebrospinal fluid samples were obtained retrospectively from normal controls (NCs; n = 27) and patients with MS (n = 40), amyotrophic lateral sclerosis (n = 22), and multiple system atrophy (n = 10) between January 1, 2008, and January 1, 2014. Patients with MS were divided into relapsing-remitting MS (RRMS; n = 30) and secondary progressive MS (n = 10). Patients with RRMS were further divided into relapse and remission groups.

Main Outcomes and Measures  The LOTUS concentration in cerebropsinal fluid was quantitatively detected by immunoblotting using a specific LOTUS antibody and the concentrations compared in accordance with the patients' clinical course, such as remission and relapse groups in RRMS and secondary progressive MS.

Results  The mean (SD) cerebrospinal fluid LOTUS concentration in the relapse group of RRMS (9.3 [3.6] µg/dL) was lower than that of NCs (19.2 [4.7] µg/dL; P < .001) whereas the level in the remission group of RRMS (19.6 [5.8] µg/dL) was similar to that of NCs. The LOTUS concentration in SPMS (6.7 [1.4] µg/dL; P < .001) was lower than that of NCs and the remission group of RRMS. The LOTUS levels in other neurodegenerative diseases, such as amyotrophic lateral sclerosis and multiple system atrophy, were normal.

Conclusions and Relevance  Variations in LOTUS concentrations were correlated with disease activity in MS. Therefore, LOTUS concentration may be useful as a possible biomarker for MS. Low LOTUS concentrations may be possibly involved in Nogo receptor-1 signaling, which may induce axonal degeneration in the relapse phase of RRMS and secondary progressive MS.

Figures in this Article

Multiple sclerosis (MS) is characterized by repeated relapses and remissions in early stages (relapsing-remitting MS [RRMS]) in most patients. Occasionally, RRMS later transforms into secondary progressive MS (SPMS), which is characterized by a persistent progressive phase without apparent relapses. Although MS is generally considered an autoimmune demyelinating disorder of the central nervous system, axonal degeneration is a recently recognized important pathological feature of RRMS.1 In particular, SPMS is characterized by widespread and prominent axonal degeneration.2 The mechanism of axonal degeneration in MS involves myelin-derived axonal growth inhibitors, such as Nogo and its receptor, Nogo receptor-1 (NgR1).36 Activation of NgR1-mediated signaling plays a substantial role in axonal degeneration in the progressive phase of MS.7 We previously identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist that prevents Nogo-NgR1 binding.8 Lateral olfactory tract usher substance is also known as cartilage acidic protein 1B. Thus, LOTUS may be an endogenous inhibitor of axonal degeneration in MS by blocking Nogo-NgR1 interactions. Here, we examined LOTUS in healthy human cerebrospinal fluid (CSF) and variations in LOTUS concentrations in the CSF of patients with MS in accordance with their clinical course. The LOTUS concentrations were also examined in CSF samples from patients with other neurodegenerative disorders.

Participants

All patients enrolled in this study underwent neurological evaluation at our hospital and were diagnosed with MS, other diseases, or were found to be normal. Samples of CSF were obtained retrospectively from 62 patients with MS, 22 patients with amyotrophic lateral sclerosis (ALS), 10 patients with multiple system atrophy (MSA), and 27 normal controls (NCs) between January 1, 2008, and January 1, 2014. All patients with MS fulfilled the McDonald criteria9 and were initially divided into the RRMS group (52 patients) or the SPMS group (10 patients). Secondary progressive MS was defined as progressive deterioration of disability for at least 6 months with an increase of at least 1 Expanded Disability Status Scale point during the last 2 years, with or without superimposed exacerbations following an initial RRMS course.10 The 52 patients of the RRMS group were further divided into a relapsing group (36 patients) or remitting group (16 patients) based on clinical findings and clinical history in medical records by trained neurologists. Twenty of the 36 patients in the relapsing group had test results that were positive on gadolinium-enhanced magnetic resonance imaging and were analyzed as patients with relapse in our study. The patients in the remitting group were defined as keeping remission state without exacerbation and relapse for at least 4 months and no abnormal lesions on gadolinium-enhanced magnetic resonance imaging based on clinical findings in the remission phase. Ten of the 16 patients clinically judged to be in a remission state met these criteria and were used in analysis. The periods of remission state in the remitting group were 4 to 44 months (15.8 months on average).

Written informed consent was obtained from each participant according to the research ethics of our hospital. This study was approved by the ethics committee of the Yokohama City University Medical Hospital.

Procedures

After the lumbar punctures, CSF samples were frozen at −80°C. Samples were centrifuged at 15 000g for 10 minutes to remove cells and debris. Samples of CSF (7.5 µL/well) were separated with sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was incubated with primary antibody against mouse LOTUS (0.25 µg/mL) for 1 hour and then incubated with horseradish peroxidase–labeled donkey antibodies against rabbit IgG (1:1000; GE Healthcare) for 1 hour. An enhanced chemiluminescence substrate was used and signals were quantified using an image analyzer (ImageQuant 400; GE Healthcare).

The Myc-His 6-tagged LOTUS protein was purified as previously described8 and determined the concentration based on a bovine serum albumin standard. Standard curves were generated by analyzing the density of the concentration of Myc-His 6-tagged LOTUS and the concentration of LOTUS in the CSF was calculated using this standard curve.

We performed the additional experiments 3 times using some of the same samples to verify intra-assay reproducibility. Furthermore, we performed additional experiments using a different kind of antibody (which is commercially available; R & D System) 3 times and under blinded conditions to determine interassay variability.

Statistical Analysis

Statistical analysis was performed with the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise analysis as appropriate, using GraphPad Prism version 6.0 (GraphPad Software). A P value less than .05 was considered to be statistically significant.

We identified LOTUS protein in human CSF with anti-LOTUS immunoblotting and confirmed LOTUS expression using peptide blocking. The patients’ characteristics in this study are summarized in the Table. We compared the CSF LOTUS concentration between NCs and patients with RRMS. The mean (SD) LOTUS concentration in patients in the relapse group of RRMS (9.3 [3.6] µg/dL) was lower than that in NCs (19.2 [4.7] µg/dL; P < .001; Figure). The LOTUS concentration in the remission group of RRMS (19.6 [5.8] µg/dL) was similar to that in NCs (19.2 [4.7] µg/dL; Figure). The LOTUS concentration in the relapse group of RRMS (9.3 [3.6] µg/dL) was lower than that in the remission group of RRMS (19.6 [5.8] µg/dL; P < .001; Figure). Intra-assay and interassay experiments yielded the same results.

Table Graphic Jump LocationTable.  Demographics and Clinical Characteristics of Patients and NCs
Place holder to copy figure label and caption
Figure.
Lateral Olfactory Tract Usher Substance (LOTUS) Concentration in Cerebrospinal Fluid (CSF)

The LOTUS concentration in the CSF of patients with relapsing-remitting multiple sclerosis (RRMS) with relapse (n = 20) and remission (n = 10), patients with secondary progressive multiple sclerosis (SPMS) (n = 10), patients with amyotrophic lateral sclerosis (ALS) (n = 22), patients with multiple system atrophy (MSA) (n = 10), and normal controls (NCs) (n = 27).

Graphic Jump Location

Next, we compared the LOTUS concentration between patients with SPMS and patients with RRMS. The LOTUS concentration in patients with SPMS (6.7 [1.4] µg/dL) was lower than that in the remission group of RRMS (19.6 [5.8] µg/dL; P < .001; Figure) and similar to that in the relapse group of RRMS (9.3 [3.6] µg/dL; Figure). No difference among the CSF LOTUS concentrations in patients with ALS, patients with MSA, and NCs was found (Figure). We found a significant difference between relapse in patients with RRMS and NCs (P < .001; Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise analysis) and between relapse and remission in RRMS (P < .001). No significant difference was found among remission in patients with RRMS, ALS, and MSA and NCs. A significant difference was found between SPMS and NCs but no significant difference was found between SPMS and relapse in RRMS. The horizontal lines represent the mean LOTUS concentration in each group and each symbol are data from an individual patient or NC.

The LOTUS concentration during the remission period was examined in the 3 patients with RRMS who maintained a remission state and recovered to NC levels following a relapse. Patient 1’s LOTUS concentration changed from 11.4 μg/dL at relapse to 16.4 μg/dL, 5 months after relapse; patient 2’s LOTUS concentration changed from 8.6 μg/dL at relapse to 14.6 μg/dL, 25 months after relapse; and patient 3’s LOTUS concentration changed from 5.1 μg/dL at relapse to 18.0 μg/dL, 26 months after relapse (eFigure in the Supplement).

Activation of NgR1 signaling or upregulation of Nogo expression in demyelinating lesions may be a main cause of axonal degeneration in the progressive phase of MS.7 We previously reported that LOTUS completely suppresses Nogo binding to NgR1 in mice.8 Accordingly, LOTUS may be an endogenous inhibitor of axonal degeneration by blocking the NgR1 function in MS. Here, the LOTUS concentration was 19.2 (4.7) µg/dL in healthy human CSF and drastically decreased in the CSF of patients with RRMS in the relapse phase (9.3 [3.6] µg/dL). A decrease in LOTUS may activate NgR1 signaling by attenuating its antagonistic action on NgR1 function and may trigger the pathological cascade that leads to axonal degeneration. However, in RRMS, a decrease in LOTUS in the relapse phase was presumably transient with minimum axonal damage and recovered to the NC level in the remission phase along with improvement in clinical findings.

Maintenance of a lower concentration of LOTUS (6.7 [1.4] µg/dL) was found in SPMS, which is characterized by a progressive clinical course and shows continuous severe axonal degeneration. We speculate that LOTUS is a possible endogenous inhibitor of axonal degeneration and that a long-term decrease in LOTUS may be 1 of the causative mechanisms for persistent axonal degeneration in the progressive phase of MS.

Furthermore, fluctuations in the CSF concentration of LOTUS may be a novel biomarker of disease activity in terms of ongoing axonal degeneration. Although magnetic resonance imaging is useful for determining a definite diagnosis or detecting relapse lesions in RRMS, the availability of this technique is generally limited in hospitals. Furthermore, conventional CSF biomarkers, such as oligoclonal bands and the IgG index, are controversial as diagnostic tools for acute relapses in MS11; the sensitivity of these biomarkers varies according to race/ethnicity and country. In European populations, more than 90% of patients with MS have positive test results for oligoclonal bands and show an increase in the IgG index while these are found in only 30% to 60% of patients with MS in Asian populations, including Japanese.12,13 Accordingly, a smaller proportion of the patients with positive oligoclonal bands and IgG Index was observed in this study cohort. Moreover, these biomarkers are not directly related to the pathogenesis of MS. Therefore, a new CSF biomarker is required for accurate diagnosis, appropriate therapy, comprehension of pathogenesis, and the development of new therapeutic strategies in MS.1 Lateral olfactory tract usher substance is a neuronal molecule8 and our data showing a good correlation between variations in LOTUS concentrations and disease dynamics strongly suggest that LOTUS may be a useful biomarker for disease activity and prognosis for both patients with RRMS and patients with SPMS.

From a clinical point of view, our findings provide new avenues for LOTUS as a useful biomarker and for understanding the pathogenesis of MS. However, our study has limitations, including the small number of patients and absence of longitudinal follow-up of CSF LOTUS levels. A large-scale longitudinal multicenter cohort study is required to confirm our results.

The CSF LOTUS concentrations in the relapse group of RRMS and SPMS were lower than that of NCs, whereas the level in the remission group of RRMS was similar to that of NCs. Variations in LOTUS concentrations were correlated with disease activity in MS. Therefore, LOTUS concentration may be useful as a possible biomarker for MS.

Corresponding Author: Kohtaro Takei, PhD, Molecular Medical Bioscience Laboratory, Department of Medical Life Science, Yokohama City University Graduate School of Medical Life Science, Suehiro-cho 1-7-29, Tsurumi-Ku, Yokohama 230-0045, Japan (kohtaro@med.yokohama-cu.ac.jp).

Accepted for Publication: October 1, 2014.

Published Online: December 1, 2014. doi:10.1001/jamaneurol.2014.3613.

Author Contributions: Drs Takei and Tanaka had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Takei.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Takahashi, Takei.

Critical revision of the manuscript for important intellectual content: Kurihara, Suzuki, Goshima, Tanaka, Takei.

Statistical analysis: Takahashi, Takei.

Obtained funding: Takahashi, Goshima, Tanaka, Takei.

Administrative, technical, or material support: Kurihara, Suzuki, Goshima, Tanaka, Takei.

Study supervision: Tanaka, Takei.

Conflict of Interest Disclosures: Research funds were obtained from the Cosmic Corporation. No other disclosures were reported.

Funding/Support: This study was supported by grants in aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Drs Takei, Tanaka, Goshima, and Suzuki); a grant for a Research and Development Project III of Yokohama City University, Japan (Drs Takei and Suzuki); a grant for medical research from the Japan Multiple Sclerosis Society (Dr Takahashi); and grants for medical research from the Takeda Science Foundation for Visionary Research and the Astellas Foundation for Research on Metabolic Disorders (Dr Takei).

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Yoko Ino, BS, Advanced Medical Research Center, Yokohama City University, and Hisashi Hirano, PhD, Yokohama City University Graduate School of Medical Life Science, for assisting with identification of LOTUS. They received no financial compensation for their contributions.

Teunissen  CE, Dijkstra  C, Polman  C.  Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet Neurol. 2005;4(1):32-41.
PubMed   |  Link to Article
Kutzelnigg  A, Lucchinetti  CF, Stadelmann  C,  et al.  Cortical demyelination and diffuse white matter injury in multiple sclerosis. Brain. 2005;128(pt 11):2705-2712.
PubMed   |  Link to Article
Karnezis  T, Mandemakers  W, McQualter  JL,  et al.  The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination. Nat Neurosci. 2004;7(7):736-744.
PubMed   |  Link to Article
Yang  Y, Liu  Y, Wei  P,  et al.  Silencing Nogo-A promotes functional recovery in demyelinating disease. Ann Neurol. 2010;67(4):498-507.
PubMed   |  Link to Article
Satoh  J, Onoue  H, Arima  K, Yamamura  T.  Nogo-A and nogo receptor expression in demyelinating lesions of multiple sclerosis. J Neuropathol Exp Neurol. 2005;64(2):129-138.
PubMed
Fournier  AE, GrandPre  T, Strittmatter  SM.  Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature. 2001;409(6818):341-346.
PubMed   |  Link to Article
Petratos  S, Ozturk  E, Azari  MF,  et al.  Limiting multiple sclerosis related axonopathy by blocking Nogo receptor and CRMP-2 phosphorylation. Brain. 2012;135(pt 6):1794-1818.
PubMed   |  Link to Article
Sato  Y, Iketani  M, Kurihara  Y,  et al.  Cartilage acidic protein-1B (LOTUS), an endogenous Nogo receptor antagonist for axon tract formation. Science. 2011;333(6043):769-773.
PubMed   |  Link to Article
Polman  CH, Reingold  SC, Banwell  B,  et al.  Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011;69(2):292-302.
PubMed   |  Link to Article
Secondary Progressive Efficacy Clinical Trial of Recombinant Interferon-Beta-1a in MS (SPECTRIMS) Study Group.  Randomized controlled trial of interferon- beta-1a in secondary progressive MS: clinical results. Neurology. 2001;56(11):1496-1504.
PubMed   |  Link to Article
Lourenco  P, Shirani  A, Saeedi  J, Oger  J, Schreiber  WE, Tremlett  H.  Oligoclonal bands and cerebrospinal fluid markers in multiple sclerosis: associations with disease course and progression. Mult Scler. 2013;19(5):577-584.
PubMed   |  Link to Article
Kira  J.  Multiple sclerosis in the Japanese population. Lancet Neurol. 2003;2(2):117-127.
PubMed   |  Link to Article
Nakashima  I, Fujihara  K, Itoyama  Y.  Oligoclonal IgG bands in Japanese multiple sclerosis patients. J Neuroimmunol. 1999;101(2):205-206.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure.
Lateral Olfactory Tract Usher Substance (LOTUS) Concentration in Cerebrospinal Fluid (CSF)

The LOTUS concentration in the CSF of patients with relapsing-remitting multiple sclerosis (RRMS) with relapse (n = 20) and remission (n = 10), patients with secondary progressive multiple sclerosis (SPMS) (n = 10), patients with amyotrophic lateral sclerosis (ALS) (n = 22), patients with multiple system atrophy (MSA) (n = 10), and normal controls (NCs) (n = 27).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Demographics and Clinical Characteristics of Patients and NCs

References

Teunissen  CE, Dijkstra  C, Polman  C.  Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet Neurol. 2005;4(1):32-41.
PubMed   |  Link to Article
Kutzelnigg  A, Lucchinetti  CF, Stadelmann  C,  et al.  Cortical demyelination and diffuse white matter injury in multiple sclerosis. Brain. 2005;128(pt 11):2705-2712.
PubMed   |  Link to Article
Karnezis  T, Mandemakers  W, McQualter  JL,  et al.  The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination. Nat Neurosci. 2004;7(7):736-744.
PubMed   |  Link to Article
Yang  Y, Liu  Y, Wei  P,  et al.  Silencing Nogo-A promotes functional recovery in demyelinating disease. Ann Neurol. 2010;67(4):498-507.
PubMed   |  Link to Article
Satoh  J, Onoue  H, Arima  K, Yamamura  T.  Nogo-A and nogo receptor expression in demyelinating lesions of multiple sclerosis. J Neuropathol Exp Neurol. 2005;64(2):129-138.
PubMed
Fournier  AE, GrandPre  T, Strittmatter  SM.  Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature. 2001;409(6818):341-346.
PubMed   |  Link to Article
Petratos  S, Ozturk  E, Azari  MF,  et al.  Limiting multiple sclerosis related axonopathy by blocking Nogo receptor and CRMP-2 phosphorylation. Brain. 2012;135(pt 6):1794-1818.
PubMed   |  Link to Article
Sato  Y, Iketani  M, Kurihara  Y,  et al.  Cartilage acidic protein-1B (LOTUS), an endogenous Nogo receptor antagonist for axon tract formation. Science. 2011;333(6043):769-773.
PubMed   |  Link to Article
Polman  CH, Reingold  SC, Banwell  B,  et al.  Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011;69(2):292-302.
PubMed   |  Link to Article
Secondary Progressive Efficacy Clinical Trial of Recombinant Interferon-Beta-1a in MS (SPECTRIMS) Study Group.  Randomized controlled trial of interferon- beta-1a in secondary progressive MS: clinical results. Neurology. 2001;56(11):1496-1504.
PubMed   |  Link to Article
Lourenco  P, Shirani  A, Saeedi  J, Oger  J, Schreiber  WE, Tremlett  H.  Oligoclonal bands and cerebrospinal fluid markers in multiple sclerosis: associations with disease course and progression. Mult Scler. 2013;19(5):577-584.
PubMed   |  Link to Article
Kira  J.  Multiple sclerosis in the Japanese population. Lancet Neurol. 2003;2(2):117-127.
PubMed   |  Link to Article
Nakashima  I, Fujihara  K, Itoyama  Y.  Oligoclonal IgG bands in Japanese multiple sclerosis patients. J Neuroimmunol. 1999;101(2):205-206.
PubMed   |  Link to Article

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eFigure. Recovery of LOTUS concentration in CSF after relapse in RRMS patients with remission.

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