# PATHOPHYSIOLOGIC INSIGHTS FROM BIOMARKER STUDIES IN NEUROLOGICAL DISORDERS

EDITED BY : Tobias Ruck, Marcello Moccia, Tobias Warnecke and Stefan Bittner PUBLISHED IN : Frontiers in Neurology and Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88963-643-3 DOI 10.3389/978-2-88963-643-3

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# PATHOPHYSIOLOGIC INSIGHTS FROM BIOMARKER STUDIES IN NEUROLOGICAL DISORDERS

Topic Editors: Tobias Ruck, University of Münster, Germany Marcello Moccia, University of Naples Federico II, Italy Tobias Warnecke, University of Münster, Germany Stefan Bittner, Johannes Gutenberg University Mainz, Germany

Citation: Ruck, T., Moccia, M., Warnecke, T., Bittner, S., eds. (2020). Pathophysiologic Insights From Biomarker Studies in Neurological Disorders. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-643-3

# Table of Contents

*07 Editorial: Pathophysiologic Insights From Biomarker Studies in Neurological Disorders*

Stefan Bittner, Marcello Moccia, Tobias Warnecke and Tobias Ruck

Boel De Paepe, Jana Zschüntzsch, Tea Šokčević, Joachim Weis, Jens Schmidt and Jan L. De Bleecker

*42 Real World Lab Data: Patterns of Lymphocyte Counts in Fingolimod Treated Patients*

Maxi Kaufmann, Rocco Haase, Undine Proschmann, Tjalf Ziemssen and Katja Akgün

*53 Real-World Lab Data in Natalizumab Treated Multiple Sclerosis Patients Up to 6 Years Long-Term Follow Up*

Maxi Kaufmann, Rocco Haase, Undine Proschmann, Tjalf Ziemssen and Katja Akgün

Rezzak Yilmaz, Antonio P. Strafella, Alice Bernard, Claudia Schulte, Lieneke van den Heuvel, Nicole Schneiderhan-Marra, Thomas Knorpp, Thomas O. Joos, Frank Leypoldt, Johanna Geritz, Clint Hansen, Sebastian Heinzel, Anja Apel, Thomas Gasser, Anthony E. Lang, Daniela Berg, Walter Maetzler and Connie Marras

*89 Quantitative EEG and Verbal Fluency in DBS Patients: Comparison of Stimulator-On and -Off Conditions*

Florian Hatz, Antonia Meyer, Anne Roesch, Ethan Taub, Ute Gschwandtner and Peter Fuhr

*96 A Smart Device System to Identify New Phenotypical Characteristics in Movement Disorders*

Julian Varghese, Stephan Niewöhner, Iñaki Soto-Rey, Stephanie Schipmann-Miletić, Nils Warneke, Tobias Warnecke and Martin Dugas

*102 Potential of Sodium MRI as a Biomarker for Neurodegeneration and Neuroinflammation in Multiple Sclerosis* Konstantin Huhn, Tobias Engelhorn, Ralf A. Linker and Armin M. Nagel

Katja Vohl, Alexander Duscha, Barbara Gisevius, Johannes Kaisler, Ralf Gold and Aiden Haghikia

*128 An Assay to Determine Mechanisms of Rapid Autoantibody-Induced Neurotransmitter Receptor Endocytosis and Vesicular Trafficking in Autoimmune Encephalitis*

Elsie Amedonu, Christoph Brenker, Sumanta Barman, Julian A. Schreiber, Sebastian Becker, Stefan Peischard, Nathalie Strutz-Seebohm, Christine Strippel, Andre Dik, Hans-Peter Hartung, Thomas Budde, Heinz Wiendl, Timo Strünker, Bernhard Wünsch, Norbert Goebels, Sven G. Meuth, Guiscard Seebohm and Nico Melzer

Katharina Birkner, Julia Loos, René Gollan, Falk Steffen, Beatrice Wasser, Tobias Ruck, Sven G. Meuth, Frauke Zipp and Stefan Bittner

Philipp Schwenkenbecher, Ulrich Wurster, Franz Felix Konen, Stefan Gingele, Kurt-Wolfram Sühs, Mike P. Wattjes, Martin Stangel and Thomas Skripuletz

*177 The Shaking Palsy of the Larynx—Potential Biomarker for Multiple System Atrophy: A Pilot Study and Literature Review*

Tobias Warnecke, Annemarie Vogel, Sigrid Ahring, Doreen Gruber, Hans-Jochen Heinze, Rainer Dziewas, Georg Ebersbach and Florin Gandor

*189 Substance P Saliva Reduction Predicts Pharyngeal Dysphagia in Parkinson's Disease*

Jens Burchard Schröder, Thomas Marian, Inga Claus, Paul Muhle, Matthias Pawlowski, Heinz Wiendl, Sonja Suntrup-Krueger, Sven G. Meuth, Rainer Dziewas, Tobias Ruck and Tobias Warnecke

Andreas Roos, Corinna Preusse, Denisa Hathazi, Hans-Hilmar Goebel and Werner Stenzel

*249 Apolipoprotein E Homozygous* e*4 Allele Status: A Deteriorating Effect on Visuospatial Working Memory and Global Brain Structure*

Janik Goltermann, Ronny Redlich, Katharina Dohm, Dario Zaremba, Jonathan Repple, Claas Kaehler, Dominik Grotegerd, Katharina Förster, Susanne Meinert, Verena Enneking, Emily Schlaghecken, Lara Fleischer, Tim Hahn, Harald Kugel, Andreas Jansen, Axel Krug, Katharina Brosch, Igor Nenadic, Simon Schmitt, Frederike Stein, Tina Meller, Dilara Yüksel, Elena Fischer, Marcella Rietschel, Stephanie H. Witt, Andreas J. Forstner, Markus M. Nöthen, Tilo Kircher, Anbupalam Thalamuthu, Bernhard T. Baune, Udo Dannlowski and Nils Opel

Andreas Johnen and Maxime Bertoux

*301 Identification of Cellular Pathogenicity Markers for SIL1 Mutations Linked to Marinesco-Sjögren Syndrome*

Christian Gatz, Denisa Hathazi, Ute Münchberg, Stephan Buchkremer, Thomas Labisch, Ben Munro, Rita Horvath, Ana Töpf, Joachim Weis and Andreas Roos

*316 Lymphocyte Count and Body Mass Index as Biomarkers of Early Treatment Response in a Multiple Sclerosis Dimethyl Fumarate-Treated Cohort*

Alessia Manni, Antonio Iaffaldano, Giuseppe Lucisano, Mariangela D'Onghia, Domenico Maria Mezzapesa, Vincenzo Felica, Pietro Iaffaldano, Maria Trojano and Damiano Paolicelli

#### *325 Dysphagia as Isolated Manifestation of Jo-1 Associated Myositis?*

Bendix Labeit, Paul Muhle, Sonja Suntrup-Krueger, Sigrid Ahring, Tobias Ruck, Rainer Dziewas and Tobias Warnecke

#### *330 Neuro-Sjögren: Peripheral Neuropathy With Limb Weakness in Sjögren's Syndrome*

Tabea Seeliger, Nils K. Prenzler, Stefan Gingele, Benjamin Seeliger, Sonja Körner, Thea Thiele, Lena Bönig, Kurt-Wolfram Sühs, Torsten Witte, Martin Stangel and Thomas Skripuletz

# Editorial: Pathophysiologic Insights From Biomarker Studies in Neurological Disorders

#### Stefan Bittner 1 , Marcello Moccia2 , Tobias Warnecke3 and Tobias Ruck 3 \*

1 Department of Neurology, Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany, 2 Multiple Sclerosis Clinical Care and Research Centre, Department of Neuroscience, Reproductive Science and Odontostomatology, Federico II University, Naples, Italy, 3 Department of Neurology with Institute of Translational Neurology, University of Münster, Münster, Germany

Keywords: biomarker, pathophysiology, neuroimmunology, neurodegeneration, pathomechanism

#### **Editorial on the Research Topic**

#### **Pathophysiologic Insights From Biomarker Studies in Neurological Disorders**

Neuroimmunological and neurodegenerative disorders are major healthcare issues, associated with high individual and socioeconomic burden. For many neurological disorders an early diagnosis is key for efficient management and therapy. However, neural tissues from brain, spinal cord, or peripheral nerves are difficult to sample, and their extraction is often associated with functional deficits. Therefore, in most cases a combination of biomarkers such as radiologic or biochemical findings are used to define a diagnosis in the clinical practice. The development of pathologicallysensitive and easy-to-measure disease biomarkers is a key factor for investigating new medications in phase II trials aiming to quickly screen their efficacy, and in phase III trials where they are coupled to clinical measures. Moreover, biomarkers are used to predict treatment efficacy and adverse event risk, paving the way for personalized medicine with individual risk assessment and prevention strategies.

In the last few decades the enormous technological progress, especially omics technologies, expanded the definition of biomarkers from biochemical and clinical, to genetic, proteomic, metabolic, or microbial markers. Interestingly, some of the biomarker studies additionally provided important insights into the pathophysiology of neurological disorders. In multiple sclerosis TNFblocking drugs can promote onset or exacerbation of MS and GWAS (genome-wide association studies) data informed about the underlying mechanisms instructing clinical practice (1). In Parkinson's disease (PD), evaluation of Lewy pathology provided new pathophysiological insights attributing PD pathology progression to a prion-like process starting in the gastrointestinal tract (2). However, many other suggested biomarkers remain solely correlative, often lacking a causative link to the underlying disease mechanisms possibly explaining their lack in sensitivity and specificity.

This Research Topic reviews current knowledge and provides new insights into pathomechanisms of a wide variety of neuroimmunological and neurodegenerative disorders from studies on (novel) risk factors and outcome predicting biomarkers.

# INFLAMMATORY DISORDERS OF THE CENTRAL NERVOUS SYSTEM

There is still an unmet need for novel, innovative biomarker approaches in different stages of central nervous system inflammation ranging from first diagnosis to evaluation of inflammatory and

#### Edited by:

Hans-Peter Hartung, Heinrich Heine University of Düsseldorf, Germany

#### Reviewed by:

Roberto Furlan, San Raffaele Hospital (IRCCS), Italy

> \*Correspondence: Tobias Ruck<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology

Received: 03 November 2019 Accepted: 14 February 2020 Published: 28 February 2020

#### Citation:

Bittner S, Moccia M, Warnecke T and Ruck T (2020) Editorial: Pathophysiologic Insights From Biomarker Studies in Neurological Disorders. Front. Neurol. 11:151. doi: 10.3389/fneur.2020.00151 neurodegenerative processes and therapy responses. In this Research Topic, Pawlitzki et al. shows the value of CSFbased biomarker approaches indicating ongoing chronic CNS inflammation even before first clinical sign of symptoms in patients with radiologically isolated syndrome, while Schwenkenbecher et al. demonstrate the importance of oligoclonal bands in the diagnostic algorithm of multiple sclerosis assessing the impact of the newest McDonald criteria. Until now, we have only a limited understanding of inflammatory and neurodegenerative processes within the CNS compartment in multiple sclerosis. Here, Birkner et al. assessed the role of ICAM-5, a CNS-restricted adhesion molecule, in mediating T cell-induced neuronal damage in the animal model of experimental autoimmune encephalomyelitis (EAE), Huhn et al. investigated the use of sodium MRI as a translational approach in MS patients. Another part of this Research Topic section focuses on the value of biomarker approaches in different real-world therapeutic settings, namely blood-based biomarkers in patients treated with fingolimod (Kaufmann et al.), dimethylfumarate (Manni et al.), and in a longitudinal setting in patients treated with natalizumab (Kaufmann et al.). The study by Vohl et al. investigates a cohort of patients treated with intrathecal corticosteroid therapy, a long-established, but sometimes under-appreciated therapeutic approach, in numerous centers specialized on the treatment of MS patients, that so far has suffered from the lack of large studies. In a cohort of 38 patients with autoimmune encephalitis (AE), Körtvelyessy et al. showed that CSF Progranulin levels were elevated during the acute phase, suggesting ongoing neuro-axonal damage, and that total-Tau (t-tau) and neurofilament light chain (NfL) levels were associated with the risk of clinical, laboratory, and radiological progression. Future studies on AE could benefit from the work of Amedonu et al., with the use of a novel assay to study molecular mechanisms of autoantibody-induced receptor internalization and to screen new molecular-based therapies.

# NEURODEGENERATION AND STROKE

Neurodegenerative diseases have been conventionally studied in relation to the genetic risk of clinical/cognitive dysfunction and neuronal damage. In line with this, Goltermann et al. showed that apolipoprotein E (APOE) ε4 homozygosity, a well-known risk factor for Alzheimer's disease (AD), was associated with worse visuospatial working memory and global brain structural alterations in 62 non-neurological subjects from the FOR2107 Marburg-Münster Affective Cohort Study.

More recently, an interplay between inflammation and neurodegeneration has been recognized. Looking at conditions that are conventionally unrelated to inflammation, Hu et al. demonstrated an inflammatory component in the normal aging and in AD, by showing an accentuated shift from T-helper (Th)- 1 to non-Th1 inflammatory pathways in the cerebrospinal fluid (CSF) (similar to what happens in multiple sclerosis). Hotter et al. evaluated 91 stroke patients not suffering from strokeassociated infections (derived from the PREDICT and 1000 Plus studies), and found that IL-6 was associated with several MRI measures of stroke severity (e.g., acute and final lesion perfusion and size), suggesting that inflammation is related to vascular parenchymal damage. Also, neuroinflammation during the fetal period could be related to neurodevelopmental disorders (e.g., autism spectrum disorder, schizophrenia). Cao et al. studied the role of α7nAChR on fetal sheep astrocytes, a classic model of fetal neurobiology, and further unraveled the complex interactions between inflammation and neurodegeneration at neurodevelopmental stages.

However, our Research Topic did not aim to underestimate clinical examination, which remains of utmost importance in the neurology clinical practice. In particular, in their review, Johnen and Bertoux suggested that the early clinical diagnosis of the behavioral variant frontotemporal dementia (bvFTD) requires an in-depth assessment of clinical signs, behavioral and psychological symptoms as well as cognitive performance. Authors strongly encouraged the design of novel neuropsychological tests covering understudied aspects of dementia (e.g., social and emotional processing, praxis abilities, interoceptive processing), and further expanding the number of clinical and neuropsychological biomarkers for the neurology clinical practice (Johnen and Bertoux).

# MOVEMENT DISORDERS, PARKINSON'S DISEASE, AND DYSPHAGIA

Over the last decade increasing scientific efforts have been made to establish biomarkers for early diagnosis of Parkinson's disease (PD) and other movement disorders. These potentially helpful biomarkers include findings from neuroimaging techniques, blood and cerebrospinal fluid (CSF) immune markers, technology-based objective measures (TOMs) provided by wearable devices, results from skin biopsies, and several others. Doppler et al. were now able to show that dermal phosphoalpha-synuclein (p-syn) in PD patients with glucocerebrosidase gene (GBA1) mutations seems to offer a similar distribution and frequency as observed in PD patients without a known mutation. Therefore, skin biopsy may be suitable to study p-syn deposition in these patients or even to identify premotor patients with GBA1 mutations (Doppler et al.). However, a panel of serum immune markers (i.e., cytokines) as analyzed using principal component analysis (PCA) was not found to allow for reliably predicting clinical PD subtypes (Yilmaz et al.). Varghese et al. introduced a technology-based system called Smart Device System (SDS) that was tested over 2 years to train an Artificial Intelligence System; this system will be further implemented for multi-modal high-resolution acceleration measurement of patients with PD or essential tremor (ET). Furthermore, Warnecke et al. used flexible endoscopic evaluation of swallowing (FEES) to detect specific pharyngolaryngeal movement disorders, such as arytenoid tremor that may help to differentiate multiple system atrophy (MSA) from PD.

In addition, biomarkers for detection of specific symptoms related to PD and other movement disorders have been studied throughout the last years. In their narrative review Prell et al. provide an overview of recent developments of bio-fluid and imaging biomarkers for dementia, depression, and fatigue in PD. They conclude that more research needs to be undertaken to find reliable combinations of predictors of these neuropsychological symptoms in PD on an individual level, and that standardization and harmonization of protocols in particular in CSF handling and neuroimaging has to be further implemented (Prell et al.). Richter et al. assessed the utility of brainstem raphe (BR) alterations evaluated by transcranial sonography (TCS) as a potential biomarker for apathy and depression in PD patients. These BR alterations correlated with depression and apathy scales indicating that a serotonergic signal disturbance might exist for both symptoms in PD (Richter et al.). Beside these neuropsychological symptoms, significant attention has being paid to gastrointestinal dysfunction and particularly swallowing disorders. Clinical research has shown that dysphagia is an often overlooked, but complex syndrome in many patients with neurodegenerative and neuromuscular disorders mainly contributing to disease burden in the majority of cases. Schröder et al. were able to show that reduced saliva substance P (SP) concentrations may predict early pharyngeal swallowing dysfunction in PD patients and may help to identify PD patients with a higher risk of developing clinically relevant dysphagia. Labeit et al. reported the first case of isolated dysphagia due to Jo-1 antibodies associated myositis. They suggest that myositisfocused diagnostics including an autoantibody panel should be done in patients with unclear dysphagia that show typical sings for myositis in the instrumental dysphagia assessment (Labeit et al.).

In the future biomarkers should not only be available for early diagnosis, but also for indicating responses to specific therapeutic interventions that help to guide treatment decisions in PD and other movements disorders. Schlenstedt et al. presented pilot data indirectly supporting the hypothesis that exercises specifically designed to target freezing of gait (FOG) might be more beneficial than non-FOG-specific interventions in PD patients. However, future studies should include larger samples and high frequency interventions to investigate the role of training balance performance to reduce the severity of FOG and to analyze which subgroup of PD patients shows the largest effect size (Schlenstedt et al.). Finally, Hatz et al. were able to demonstrate that deep brain stimulation of the subthalamic nucleus (STN) used to treat motor complications in advanced PD does not alter verbal fluency performance in a systematic way at group level. Nevertheless, when STN stimulation produces an alteration of verbal fluency performance in an individual PD patient, it was inversely correlated with left temporal delta power as measured by quantitative EEG (Hatz et al.).

# DISORDERS OF THE PERIPHERAL NERVOUS SYSTEM

The peripheral nervous system (PNS) consists of nervous structures (cranial and spinal nerves, enteric nervous system) and their associated receptors and organs. The diagnosis of PNS disorders is a multi-step process and often complicated by unspecific findings. Instructive cellular and non-cellular biomarkers for diagnosis, prognosis and/or treatment management are largely missing. However, recent years including studies of this Research Topic support the high relevance of biomarkers for an adequate disease management. Correspondingly, Stuhlmüller et al. review the role of myositis-specific and -associated antibodies in idiopathic inflammatory myopathies (IIM). They highlight their value in the diagnostic process and prognosis (organ involvement, risk for malignancies), and elaborate on detection methods and associated clinical phenotypes (Stuhlmüller et al.). De Paepe et al. demonstrate an upregulation of osmolyte accumulators and identify associated inflammatory signaling pathways in skeletal muscle cells upon pro-inflammatory stimuli in vitro and in skeletal muscle biopsies of IIM patients. The authors characterize specific osmolyte accumulators as potential biomarkers for regenerating muscle fibers or autoreactive immune cells (De Paepe et al.).

However, biomarker research might not only unravel associations with clinical parameters but also reveal important insights into pathophysiological disease mechanisms. With an unbiased proteomic profiling Roos et al. identified key molecules involved in the pathogenesis of sporadic inclusion body myositis (sIBM), which were further characterized by qPCR, immunostaining, immunofluorescence in situ and in silico studies. By these means, they were able to demonstrate a key role of specific macrophage subtypes and molecules (CD74, CD163, and STAT1) involved in their activation and type I interferon and interferon gamma associated pathways in sIBM (Roos et al.). With a similar proteomic approach Kölbel et al. identified candidate protein biomarkers in skeletal muscle of laminin-211 deficient type 1 A (MDC1A) congenital muscular dystrophy (CMD), one of the most devastating CMDs characterized by severe muscle weakness, brain abnormalities, and delayed motor milestones. The expression of 86 proteins associated with fibrosis and altered synaptic transmission was specifically linked to MDC1A skeletal muscle. Moreover, the observed alterations in metabolic pathways and mitochondrial function might identify new therapeutic targets (Kölbel et al.).

Changes in protein function, folding or metabolism is a common feature of several degenerative disorders of the PNS. In Marinesco-Sjögren syndrome (MSS) characterized by a clinical triad of bilateral cataracts, ataxia, and myopathy, mutations in the SIL1 gene lead to protein misfolding. However, read-out systems to identify pathogenic variants of SIL1 mutations are missing. Therefore, Gatz et al. introduce an in vitro system identifying the pathogenic consequences of different mutations on a cellular level. In the future, these findings can instruct genetic testing of patients with suspected MSS.

Besides disorders primarily affecting structures of the PNS, systemic autoimmune diseases such as Sjögren's syndrome might involve the PNS as well. In this Research Topic, Seeliger et al. provide a cross-sectional study of 144 patients with polyneuropathy of whom 44 were diagnosed with Sjögren's syndrome. They describe clinical findings and discuss potential tools to identify Neuro-Sjögren (Seeliger et al.). Other systemic inflammatory disorders such as vasculitides can also affect structures of the nervous system including the PNS resulting in a variety of potential symptoms. Strunk et al. review potential biomarkers in the context of nervous system vasculitides with a focus on body fluid derived markers and discuss their diagnostic and prognostic value.

Overall, the current Research Topic provides a deeper understanding of the pathomechanisms underlying neurological disorders and identifies new biomarkers and their pathophysiologic background. Thereby, the Research Topic

#### REFERENCES

might initiate hypothesis-driven refinement of current and novel biomarkers.

## AUTHOR CONTRIBUTIONS

SB, MM, TW, and TR edited the Research Topic and drafted and revised the editorial manuscript. All authors read and approved the final version of the manuscript.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Bittner, Moccia, Warnecke and Ruck. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Brainstem Raphe Alterations in TCS: A Biomarker for Depression and Apathy in Parkinson's Disease Patients

Daniel Richter 1 , Dirk Woitalla1,2, Siegfried Muhlack 1 , Ralf Gold1,3, Lars Tönges 1,3 and Christos Krogias 1 \*

1 Department of Neurology, St. Josef-Hospital, Ruhr University Bochum, Bochum, Germany, 2 Department of Neurology, Katholische Kliniken Ruhrhalbinsel, Essen, Germany, 3 Neurodegeneration Research, Protein Research Unit Ruhr (PURE), Ruhr University Bochum, Bochum, Germany

#### Edited by:

Tobias Warnecke, University Hospital Muenster, Germany

#### Reviewed by:

Georgios Tsivgoulis, National and Kapodistrian University of Athens, Greece Matteo Bologna, Sapienza Università di Roma, Italy

> \*Correspondence: Christos Krogias<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurology

Received: 19 May 2018 Accepted: 18 July 2018 Published: 07 August 2018

#### Citation:

Richter D, Woitalla D, Muhlack S, Gold R, Tönges L and Krogias C (2018) Brainstem Raphe Alterations in TCS: A Biomarker for Depression and Apathy in Parkinson's Disease Patients. Front. Neurol. 9:645. doi: 10.3389/fneur.2018.00645 Depression and apathy can both be present in patients with Parkinson's disease (PD) while e. g., essential tremor (ET) patients mostly only report depressive symptoms. In PD, depression has been linked with brainstem raphe (BR) signal alterations in transcranial sonography (TCS) but apathy has not been evaluated in such terms as a putative biomarker. Furthermore, the BR has only been investigated using a singular axial TCS examination plane, although coronal TCS examination allows a much more accurate evaluation of the craniocaudal formation of serotonergic raphe structures in the midbrain area. The objective of this study was to investigate the value of coronal TCS examination for the detection of BR signal alterations and clinically correlate it to apathy in patients with PD, ET and healthy controls (HC). We prospectively included PD patients (n = 31), ET patients (n = 16), and HC (n = 16). All were examined by TCS in the axial and coronal plane with focus on BR signal alterations. LARS and BDI-II scores were conducted to assess apathic and depressive symptoms in the study population. In a detailed analysis we found that the correlation of coronal and axial TCS alterations of BR was very high (rho = 0.950, p < 0.001). BR signal alterations were more frequent in PD patients than in ET patients and HC, while it was not different between ET patients and HC. In the PD patient group, BDI-II and LARS scores were negatively correlated to BR signal changes in TCS in a significant manner (BDI-II and axial BR: p = 0.019; BDI-II and coronal BR: p = 0.011; LARS and axial BR: p = 0.017; LARS and coronal BR: p = 0.023). Together in this brainstem ultrasound study we find a significant association of BR signal alterations with clinically evident apathy and depression in patients with PD. Therefore, TCS might enable the identification of a subgroup of PD patients which are at higher risk to suffer from or to develop depression or apathy.

Keywords: sonography, TCS, raphe, apathy, depression

#### INTRODUCTION

Transcranial sonography (TCS) is increasingly applied for the differential diagnosis of Parkinson's disease (PD) patients. Hyperechogenicity of substantia nigra (SN) has been identified as a biomarker and risk factor for PD patients and thus has been included as a diagnostic item in the new MDS research criteria for prodromal PD (1).

Depression and apathy frequently appear in PD patients and can represent early non-motor symptoms (2–4). Several studies have shown that many PD patients are affected by depression, even in the prodromal state of disease (5). Apathy has been described to appear independently from depression (6) and can severely impact quality of life (7). Brainstem raphe (BR) alterations in TCS have been associated with depressed PD patients (8, 9) but were not observed in patients with essential tremor (ET). So far, BR signal alterations have never been examined in relation to apathy and, furthermore, have only been evaluated using a singular axial TCS plane in spite of an additional coronal examination (10).

In this study, we investigated symptoms of apathy and depression by using the Lille apathy rating scale (LARS) and the Beck Depression Inventory II (BDI-II) in PD patients, ET patients and healthy controls. Recently, a new coronal examination plane has been introduced in the field of TCS by our group in order to study Substantia nigra echogogenicity (10). We applied this methodology of an additional coronal TCS examination now for BR alterations and evaluated this finding as a putative biomarker for depression and apathy in PD.

#### MATERIALS AND METHODS

#### Subjects

We prospectively included patients fulfilling the clinical diagnostic criteria of the UK Parkinson's Disease Society Brain Bank for idiopathic PD (11) or patients meeting the diagnostic criteria for ET (12). Participants with an insufficient transtemporal bone window were excluded. All patients received best medical treatment according to international guidelines for the treatment of advanced Parkinson's disease (13) or essential tremor (14). None of the participants of this study received anti-depressive therapy. Additionally, healthy controls (HC) were included to serve as an age-matched control group. The same clinical assessment and TCS imaging was performed for every subject in this study.

All subjects were included after detailed information about the study and gave written informed consent. In accordance to the Helsinki Declaration of 1975, the study was approved by the local university ethics committee of the Ruhr University Bochum, Germany (approval no. 4961-14).

#### Clinical Assessment

All PD and ET patients as well as the group of HC underwent clinical neurological assessment, done by a trained and experienced interviewer. Demographic and clinical data was assessed including disease duration and comorbidity (**Table 1**).

To evaluate motor symptoms of PD, part III of the Unified Parkinson's Disease Rating Scale (UPDRS) (15) was conducted in the entire study cohort. Definition of depression and apathy was based on established scores: using the Beck Depression Inventory II (16) to investigate severity of depression symptoms. A score of ≥19 points was defined as cut-off value for depression (17). Using the Lille Apathy Rating Scale (LARS) (18) to assess apathy, a cutoff-score of ≥-16 was chosen for apathy, since this cut-off-value has shown a high sensitivity of 87% and a high specificity of 93% in diagnosing apathy (18).

#### Transcranial Sonography

Transcranial sonographic (TCS) examination was performed on the same day as clinical assessment, and was performed by two experienced investigators (DR, CK) being blinded to clinical scores using a phased array ultrasound system equipped with a 2.5-MHz transducer (Aplio R XG Ultrasound System, Toshiba Medicals, Tochigi, Japan). A penetration depth of 150 mm and a dynamic range of 45–50 dB were chosen. Image brightness and time gain compensations were adapted as needed for each examination.

The examination protocol of the axial examination plane was based on previous published recommendations for TCS (19). Using the transtemporal approach, the midbrain and diencephalic examination planes were visualized in axial section. To evaluate SN hyperechogenicity, a planimetric measurement of SN was performed. According to previously published studies (20), we used the mean + 1 SD of SN echogenic area in the group of HC for each plane as upper normal value defining SN hyperechogenicty. In general, an enlarged echogenic area of the SN is a predictive biomarker with high sensitivity and specificity for the diagnosis of PD (10).

The echogenicity of brainstem raphe (BR) was classified semiquantitatively on a three-point scale: 0 = raphe structure not visible, 1 = slight and interrupted echogenic raphe structure, 2 = normal echogenicity (echogenicity of raphe structure is not interrupted) (**Figure 1**). Different to the enlarged echogenic area of the SN in PD, a reduced echogenic signal of the BR is thought to visualize changes in the serotonergic system (9).

Similar to the axial examination plane protocol, the temporal bone window was used to assess also the coronal TCS plane: by rotating the transducer for 90◦ , the hypoechogenic midbrain is visualized in longitudinal view including SN and BR (**Figure 2**).

The sonographic findings were stored, so that the investigators could perform a second evaluation and classification of the results from the other investigator. In the case of discrepant ratings, a consensus was accomplished, subsequently.

#### Statistical Analysis

The descriptive statistics are given as median and interquartile range throughout the manuscript. As needed, the range is given additionally. Using appropriate nonparametric tests, the groups were statistically compared and correlation analysis were performed (Kruskal-Wallis H test, Mann-Whitney–U test,

#### TABLE 1 | Demographic, clinical and sonographic data.

Boldface p-values indicate statistical sgnificance. Values are given in median and (interquartile range). MDR-UPDRS, Part 3 of the Movement Disorder Society Unified Parkinson Disease Rating Scale, BDI-II, Becks Depression Inventory II; LARS, Lille Apathy Rating Scale; TCS, Transcranial sonography.

aSignificant difference between the three groups but not between ET patients and HC; bSignificant difference between the three groups and also between ET patients and HC; cSignificant difference between the three groups but not between PD and ET patients; HKruskal-Wallis H test; UMann-Whitney-U test;

F Freeman-Halton extension of Fisher's exact test.

Wilcoxon-Test, Fisher's exact test, Freeman-Halton extension of Fisher's exact test, Spearman rho analysis) with SPSS 23.0 for Mac.

#### RESULTS

#### Study Population and Clinical Features

We screened 36 patients with Parkinson's disease; four patients (11.1%) were excluded owing to an inadequate temporal bone window and one patient (2.7%) was excluded owing to severe dementia. Thus, 31 PD-patients (21 men, 10 women; age = 69.0 [14.0] years) were included into the study. Furthermore, one of the initially 17 patients with essential Tremor was excluded owing to an inadequate temporal bone window resulting in 16 ET patients (8 men, 8 women; age = 63.5 [21.25] years) being included. As control group, 16 healthy subjects were recruited (6 men, 10 women; age = 58.0 [9.25] years). LARS could be performed in 44 participants of the patient population. Detailed information about the clinical characteristics of all participants is demonstrated in **Table 1**.

#### Symptoms of Depression and Apathy

Prevalence of depression based on BDI-II definition value differed significantly over the three groups. Owing to the definition, 10 (32.3%) PD patients, 3 (18.8%) ET patients and none of the HC were depressive in our investigation (Freeman-Halton extension of Fisher's exact test, p = 0.024). Additionally, there was a strong tendency for difference between PD-patients, ET-patients and HC regarding to the BDI-II Score (Kruskal-Wallis H test, p = 0.05). BDI-II scores of PD-patients and

FIGURE 2 | Corresponding MRI and TCS images of coronal examination planes in a patient with idiopathic Parkinson's disease. Coronal MR scanning plane (A) and corresponding TCS examination plane (B) at brainstem level. (C) Zoom in of midbrain structures for planimetric assessment of the echogenic area of substantia nigra and evaluation of brainstem raphe. The large arrows in (A) and (B) indicate substantia nigra (note that in TCS the SN is displayed echogenic). White asterisks mark the third ventricle. Arrowheads in (A) and (B) indicate the inferior horn of lateral ventricle. Small arrow in (C) indicates the normal appearing brainstem raphe. TL, temporal lobe; P, Pons; T, thalamus.

ET-patients showed comparable results (Mann-Whitney–U test p = 0.770). The BDI-II score was significantly different between PD-patients and HC (Mann-Whitney–U test, p = 0.02) while the BDI-II scores of ET-patients and HC did not show a substantial difference as the postulated p-value level of statistical significance could not be reached (Mann-Whitney–U test, p = 0.056).

Analyzing the appearance of apathy, 12 (38.7%) PD patients, one (7.7%) ET patient and none of the HC were apathic indicating a significant difference in apathy prevalence over the groups (Freeman-Halton extension of Fisher's exact test, p = 0.002). Between ET patients and HC no difference in apathy prevalence was found (Fisher's exact test, p = 1.000). Regarding to LARS scores, there was also significant difference over the three groups (Kruskal-Wallis H test, p < 0.001). This difference in LARS scores appeared significant between PD and ET patients (Mann-Whitney–U test, p = 0.023) and significant between ET patients and HC (Mann-Whitney–U test, p = 0.045) and PD patients and HC (Mann-Whitney–U test, p < 0.001), respectively.

In respect to coexisting apathy and depression, 5 (41.6%) of the apathic PD patients had only apathy and 7 (58.3%) had apathy and depression. Concerning depressive PD patients, 3 (30.0%) had only depression and 7 (70.0%) had both depression and apathy. Interestingly, in these subgroups of PD patients with isolated either depressive (3 patients, 9.7%) or apathic (5 patients, 16.1%) symptoms, only one (3.2%) PD patient with isolated apathy showed an altered BR Signal in TCS.

#### Correlation of LARS and BDI-II

For each group of the study population BDI-II and LARS scores were correlated. Only for PD patients a significant positive correlation was found (PD: rho = 0.657, p < 0.001). In ET patients and in the group of HC, no significant correlation was determined (ET: rho = 0.075, p = 0.808; HC: rho = 0.131, p = 0.630).

#### Transcranial Sonography Findings

In one ET patient the BR could not sufficiently be evaluated. In one HC only the axial sonographic BR analyzes could be examined. In both cases there was a partially insufficient bone window. Evaluation of BR echogenicity revealed that 10 (32.3%) of the 31 patients with PD, but only one (6.7%) of the remaining 15 patients with ET and only one (6.3%) of the 16 healthy controls exhibited a reduced BR signal in the axial TCS plane indicating structural alterations in this area (Freeman-Halton extension of Fisher's exact test, p = 0.044). Using the coronal TCS plane, almost the same distribution of BR alterations in the groups was found (Freeman-Halton extension of Fisher's exact test, p = 0.119). Correlation of axial and coronal TCS results in BR region was very high (rho = 0.950, p < 0.001) (**Table 1**).

As BR signal alterations in TCS were very rare in ET patients und HC, correlation analysis of BDI-II, LARS and the sonographic BR results was only performed in the PD group determining a significant negative correlation between BDI-II and BR (rho = −0.420, p = 0.019) and LARS and BR (rho = −0.427, p = 0.017) using the axial TCS plane. Assessing BR through the coronal TCS plane in the PD population, the negative correlation values between BDI-II and BR (rho = −0.434, p = 0.015) and LARS and BR (rho = −0.407, p = 0.023) were comparable to the axial TCS assessment. The correlation analysis is also visualized as scatterplots in **Figure 3**.

Comparing BDI-II and LARS scores between PD patients with and without BR signal alterations in TCS, a significant difference was found. PD patients with BR alterations in axial TCS plane had higher scores in BDI-II (Mann-Whitney–U test, p = 0.025) and LARS (Mann-Whitney–U test, p = 0.022) compared to PD patients without BR signal changes. The same relation was found for coronal TCS plane by comparing the BDI-II and LARS scores in PD patients and without BR signal changes (BDI-II: Mann-Whitney–U test, p = 0.029; LARS: Mann-Whitney–U test, p = 0.018). Findings are summarized in **Table 2**.

No differences in BDI-II scores were found if depressive PD patients with and without BR signal alteration in TCS were compared irrespective of applying the axial (Kruskal Wallis H Test, p = 0.469) or coronal TCS plane (Mann-Whitney–U test, p = 0.257). Moreover, no differences in LARS scores were found, comparing apathic PD patients with and without BR signal alteration in TCS, neither for the axial (Kruskal Wallis H Test, p = 0.683) nor for the coronal TCS plane (Mann-Whitney–U test, p = 0.792).

Concerning SN hyperechogenicity, the mean ± 1 SD in the group of HC was calculated as 0.19 cm2 for axial TCS plane resulting in a robust cut-off value for SN hyperechogenicity of unilateral echogenic SN area of at least 0.20 cm2 .

28 PD patients (90.3%), three HC (18.8%) and two ET patients (12.5%) showed SN hyperechogenicity (Freemann-Halton extension of Fisher's exact test, p < 0.001). Relating to the appearance of axial SN hyperechogenicity in the ET patients compared with the HC, no difference between these groups was found (Fisher's exact test, p = 1.000).

In the group of PD patients, the area of the SN in TCS was significantly larger than the SN area of the ET patients and HC (Kruskal-Wallis H test, p < 0.001), respectively. No difference in SN areas was found between ET patients and HC (Mann-Whitney–U test, p = 1.000).

#### DISCUSSION

To our knowledge, this is the first study to correlate BR signal alterations with symptoms of apathy in PD patients. Furthermore, this is the first study which detects BR echogenicity additionally in the recently introduced coronal TCS examination plane. We could show, that BR signal alterations detected by axial TCS examination plane appear more frequently in PD patients compared to ET patients and HC. Similarly, concerning the coronal TCS examination plane, we could confirm a higher prevalence of BR signal alterations in PD patients compared to ET patients and HC. With regard to BR signal changes, the correlation of axial and coronal TCS examination planes was very high indicating no superiority of one TCS plane compared to the other. However, as sonographic raphe analysis is based on a three-point scale we did not expect a substantial difference between the results of both TCS planes a priori.

In other studies, there have been discrepancies in the prevalence of apathy and its status as an independent symptom

#### TABLE 2 | Relation of BR, LARS, and BDI-II.

Boldface p-values indicate statistical significance. Values are given in median and (interquartile range). BDI-II, Becks Depression Inventory II; LARS, Lille Apathy Rating Scale; TCS, Transcranial sonography; BR, Brainstem raphe; BR–, With brainstem raphe alterations; BR+, Without brainstem raphe alterations; UMann-Whitney-U test.

of PD. Brok et al. found that about 40% of PD patients are suffering from apathy (21). In our study, we found comparable results by determining apathy in about 38.7% of our PD patient population. We could show that a higher grade of apathy was negatively correlated to BR signal alterations in TCS and that PD patients with BR signal changes in TCS had higher scores in LARS. BR alterations in TCS might appear as a structural correlate to apathic symptoms and thus could serve as a correlating biomarker for apathy in PD. Furthermore, TCS might allow to identify PD patients which are at high risk of developing apathy. To validate these findings, a prospective longitudinal study paradigm is needed.

One of the ET patients and none of the HC were apathic according to the definition. Apathy has been occasionally described to occur independently from depression in ET patients (22). In our study, ET patients had higher overall scores in LARS compared to HC indicating a difference in the magnitude of apathic symptoms between these two groups.

In our study, PD patients with BR signal alterations showed significantly higher scores in BDI-II compared to those without BR signal alterations. Independently of the applied TCS plane, the BR signal alteration in TCS was significantly negatively correlated to the BDI-II scores in the PD patients group. This suggests that a structural change in the BR could be associated with the occurrence of depression as well as the severity of depressive symptoms in PD patients and thus might also have a value as a biomarker for depression in PD. Therefore, BR evaluation by TCS might allow to identify a subpopulation of PD patients which are at higher risk to develop a depressive symptomatology and would benefit from a more frequent follow up or more intensive medical treatment. Additionally, we have found a correlation of BR signal changes and apathic symptoms indicating that BR alterations in PD could serve as a biomarker for both apathy and depression. In most of the PD patients apathy and depression were present simultaneously and BDI-II and LARS scores were significantly positively correlated indicating a coexistence of apathic and depressive symptoms in PD.

A reduced BR signal, seen as absent or interrupted echogenic midline structure, is thought to reflect alterations in the serotonergic system and has been reported to be associated with depression in PD patients and with other extrapyramidal neuropsychiatric disorders like Huntington's disease (8, 9, 23). Anatomically, the echogenic midline in general represents various nuclei and fiber tracts of which the dorsal raphe nucleus is one of the main structures. The raphe nucleus is the major origin for serotonin release in the brain (24). A reduced echogenic signal of the BR could be due to alterations in the micro-architecture of this region (25, 26) and thus reflect a serotonin deficiency which is involved in depression pathophysiology.

The pathophysiological basis of apathy is still unknown. Concerning PD, this study found a correlation between sonographic BR alteration and apathy. As the echogenic raphe midline has much more fine anatomical structures than the raphe nuclei, it is also possible that microanatomical changes in other brain regions involved in the serotonin metabolism exist in addition. To our knowledge, sonographic BR signal alterations in PD have only been associated to mood disorders. Interestingly, a recent study has found a significant correlation between tremor severity in PD and raphe dysfunction measured by 123 ioflupanefluoropropyl-carbomethoxy-3-beta-4-iodophenyltropane single photon emission computed tomography images (27). Further studies should check if sonographic BR signal changes are also linked to motor symptoms in PD.

Because the current BR evaluation in TCS is limited to a three-point scale, the discrimination of BR assessment might not be precise enough to detect differences in the severity of depression. We could not correlate the severity of depression in depressive PD patients to the extent of BR alterations in our study.

Interestingly, no difference with regard to BR signal alterations was found between ET patients and HC, although depression was present in some cases and BDI-II scores were higher in ET patients compared to HC as has been shown before (28, 29). This implies that depression in ET in contrast to PD does not seem to be associated with BR alterations in TCS.

In conclusion, BR signal alterations were found more frequently in PD patients compared to ET patients and HC independent of the applied axial or coronal TCS examination plane. BDI-II and LARS scores of PD patients could be correlated to BR signal alterations of both TCS planes. This indicates that a serotonergic signal disturbance might exist for both depression and apathy and that pharmacologic therapy should take these findings into account. In the case of depression, it has been already reported that a reduced BR signal might be associated with better treatment responses to serotonin-reuptake-inhibitors (SSRI) (30). Further prospective and longitudinal studies are urgently needed to validate these study results in PD

#### REFERENCES

in order to substantiate this novel tool to more precisely identify PD and ET patients at risk to develop depression and apathy.

#### AUTHOR CONTRIBUTIONS

DR contributed to conceptualizing, organizing the project, analyzing, acquiring, and interpreting the data and writing of the first draft. DW contributed to conceptualizing and revising the manuscript. SM and RG contributed to conceptualizing, organizing the project and revising the manuscript. LT contributed to organizing the project, analyzing, acquiring and interpreting the data and revising the manuscript. CK contributed to conceptualizing, organizing the project, analyzing, acquiring and interpreting the data and revising the manuscript.

societies and the movement disorder society-european section. Eur J Neurol. (2006) 13:1186–202. doi: 10.1111/j.1468-1331.2006.01548.x

**Conflict of Interest Statement:** RG has received consultation fees and speaker's honoraria from BayerSchering, BiogenIdec, MerckSerono, Novartis, Sanofi-Aventis and TEVA. He also acknowledges grant support from BayerSchering, BiogenIdec, MerckSerono, Sanofi-Aventis and TEVA, none related to this manuscript. LT served as a member of AbbVieParkinson Advisory Board. CK received Honoraria for oral presentations or travel grants for scientific meetings from Bayer Vital, Bristol-Meyer Squidd, and Boehringer Ingelheim, none related to this manuscript.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Richter, Woitalla, Muhlack, Gold, Tönges and Krogias. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Biomarkers of Neurodegeneration in Autoimmune-Mediated Encephalitis

Peter Körtvelyessy 1,2,3,4 \*, Harald Prüss 3,4, Lorenz Thurner 5 , Walter Maetzler 6,7,8 , Deborah Vittore-Welliong9 , Jörg Schultze-Amberger 10, Hans-Jochen Heinze1,2,11 , Dirk Reinhold12, Frank Leypoldt 8 , Stephan Schreiber 13 and Daniel Bittner 1,2

1 Department of Neurology, University Hospital Magdeburg, Magdeburg, Germany, 2 German Center for Neurodegenerative Diseases Magdeburg, Magdeburg, Germany, 3 Department of Neurology, Charité-Universitätsmedizin Berlin, Berlin, Germany, 4 German Center for Neurodegenerative Diseases Berlin, Berlin, Germany, 5 José Carreras Center for Immuno- and Gene Therapy and Internal Medicine I, Saarland University Medical School, Homburg, Germany, 6 Department of Neurodegeneration, Hertie Institute for Clinical Brain Research (HIH), University of Tübingen, Tübingen, Germany, 7 German Center for Neurodegenerative Diseases Tübingen, Tübingen, Germany, 8 Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany, 9 Department of Neurology and Epileptology, Universitätsklinikum Tübingen, Universität Tübingen, Tübingen, Germany, 10 Department of Neurology, Median Clinic Kladow, Kladow, Germany, 11 Department of Behavioral Neurology, Leibniz Institute for Neurobiology, Magdeburg, Germany, 12 Department of Immunohistopathology, Institute of Molecular and Clinical Immunology, Magdeburg, Germany, 13 Asklepios Department of Neurology, Brandenburg a.d. Havel, Germany

#### Edited by:

Stefan Bittner, Johannes Gutenberg-Universität Mainz, Germany

#### Reviewed by:

Nico Melzer, Universität Münster, Germany Michael R. Pranzatelli, National Pediatric Neuroinflammation Organization, Inc., United States

\*Correspondence: Peter Körtvelyessy<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology

Received: 12 May 2018 Accepted: 25 July 2018 Published: 19 September 2018

#### Citation:

Körtvelyessy P, Prüss H, Thurner L, Maetzler W, Vittore-Welliong D, Schultze-Amberger J, Heinze H-J, Reinhold D, Leypoldt F, Schreiber S and Bittner D (2018) Biomarkers of Neurodegeneration in Autoimmune-Mediated Encephalitis. Front. Neurol. 9:668. doi: 10.3389/fneur.2018.00668 Progranulin (PGRN), Total-Tau (t-tau), and Neurofilament light chain (NfL) are well known biomarkers of neurodegeneration. The objective of the present study was to investigate whether these parameters represent also biomarkers in autoimmune-mediated Encephalitis (AE) and may give us insights into the pathomechanisms of AE. We retrospectively examined the concentration of PGRN in the cerebrospinal fluid (CSF) and serum of 38 patients suffering from AE in acute phase and/or under treatment. This AE cohort comprises patients with autoantibodies against: NMDAR (n = 18 patients), Caspr2 (n = 8), Lgi-1 (n = 10), GABAB(R) (n = 1), and AMPAR (n = 1). Additionally, the concentrations of NfL (n = 25) and t-tau (n = 13) in CSF were measured when possible. Follow up data including MRI were available in 13 patients. Several age-matched cohorts with neurological diseases besides neuroinflammation or neurodegeneration served as control groups. We observed that PGRN was significantly elevated in the CSF of patients with NMDAR-AE in the acute phase, but normalized at follow up under treatment (p < 0.01). In the CSF of other patients with AE PGRN was in the range of the CSF levels of control groups. T-tau was highly elevated in the CSF of patients with temporal FLAIR-signal in the MRI and in patients developing a hippocampal sclerosis. NfL was exceptionally high initially in Patients with AE with a paraneoplastic or parainfectious cause and also normalized under treatment. The normalizations of all biomarkers were mirrored in an improvement on the modified Rankin scale. The data suggest that the concentration of PGRN in CSF might be a biomarker for acute NMDAR-AE. Pathological high t-tau levels may indicate a risk for hippocampal sclerosis. The biomarker properties of NfL remain unclear since the levels decrease under treatment, but it could not predict severity of disease in this small cohort. According to our results, we recommend to measure in clinical practice PGRN and t-tau in the CSF of patients with AE.

Keywords: progranulin, neurofilament light chain, NMDAR encephalitis, Lgi-1 encephalitis, Caspr2 encephalitis, tau, autoimmune encephalitis

**19**

# INTRODUCTION

Since the appearance of antibody-mediated autoimmune encephalopathy (AE) numerous antibodies (ab) have been linked to different clinical symptoms such as limbic encephalitis, faciobrachial dystonic seizures or dementia-like symptoms (1–3). Biomarkers of neurodegeneration mirror certain pathomechanisms of neuronal or axonal loss. The measurement of these biomarkers should bear the potential to provide useful information in everyday clinical life, e.g., to monitor the immunosuppressive therapy in Patients with AE. CSF antibody titres in e.g., contactin-associated-protein-receptor-2 (Caspr2)- AE or Leucin-rich glioma inactivated ptotein-1 (Lgi-1)-AE do not mirror the disease course in a linear way (4, 5). Furthermore, the clinical course in several patients suggests that an antibody titer independent pathomechanism might take place (6–8). The underlying mechanisms causing this dichotomy of clinical symptoms and antibody titer are largely unknown (8). One possible explanation could be the effect of the long survival of plasma cells in the brain (9). The brain-resident plasma cells itself cannot be measured as yet, but the damage possibly caused by autoantibodies should be detected via biomarkers for neuronal and axonal loss such as t-tau, PGRN, and NfL.

Recently, a direct connection between neurodegenerative mechanisms and AE has been detected in AEs mediated by IgLON5 causing an atypical tauopathy (10). Vice versa a correlation between autoimmune diseases and Tar DNA-binding Protein 43 (TDP-43) mediated neurodegeneration in FTD patients has been reported (11). There is also some debate about IgA-NMDAR-Abs and IgM-NMDAR-Abs (3, 12, 13) causing dementia-like symptoms and mimicking neurodegenerative diseases. Histopathological examinations in patients with AE have been focused on the immunological mechanisms triggered and maintained by the antibodies (8) disregarding a systematic research for markers of neurodegeneration so far. Only one case report of a Lgi-1 antibody positive patient presenting some neurodegenerative markers has been reported at autopsy, without witnessing pathological changes in alpha-synuclein, beta-amyloid, or neurofibrillary tangle (14). MRI findings and long-term neuropsychological data also suggest an involvement of the frontal and temporal lobes in the clinical course of the NMDAR-AE and voltage-gated-potassium channel (VGKC) complex-mediated AE (15–18). Another group has looked at the glial fibrillary acid, NfL and t-tau levels in patients with suspected AE (19). Their group only encompassed four patients with NMDAR-AE and one Lgi-1 patient with most of them having a status epilepticus (SE) before, as SE is known for confounding the protein levels in the CSF (20, 21). They found higher NfL and t-tau levels in all patients, which is most likely due to the SE before. In pediatric opsoclonus-myoclonus syndrome caused by antibodies with intracellular epitopes, immunosuppressive treatment has shown to decrease the CSF-Neurofilament light chain levels together with a concomitant clinical improvement (22, 23).

Here, we examined concentrations of PGRN, NfL, and ttau, well-established biomarkers of neurodegeneration, in CSF and serum of 38 patients with antibody positive AE. The aim of this study is to investigate if these proteins are possible biomarkers in Patients with AE. Also, the knowledge about the biomarkers of neurodegeneration CSF-levels may give clues about the pathological mechanisms in these patients.

# METHODS

#### Clinical Cohort

This retrospective study was performed according to the local ethical committees in Berlin, Potsdam, Brandenburg, Magdeburg and Bielefeld, respectively. All patients gave written and informed consent (ethics committee approval number 100/16). We included only patients with a proven AE by clinical symptoms as recommended by Graus et al. (24) and detection of pathological antibodies with extracellular epitopes via indirect immunofluorescence tests. The samples of AE-Patients were collected from April 2013 until October 2017 in Berlin, Potsdam, Tübingen, Bielefeld, and Magdeburg where their samples were initially stored at −80◦C and sent to Magdeburg. Every sample was stored in Magdeburg at −80◦C and all biomarkers were measured in Magdeburg. All samples were run in duplicate with the mean taken as result. Samples were measured over time and not in a batch.

All patients received a lumbar puncture as part of the diagnostical work up when presenting for the first time on the ward and for antibody titre control in follow up depending on each individual disease course. At least 5 ml up to 13 ml CSF was taken and serum was collected in serum separator tubes and centrifuged at site. Every patient (n = 38) suffered from a limbic encephalitis including its variants, e.g., limbic encephalitis together with vegetative and/or peripheral neurological symptoms. All patients improved under immunosuppressive therapy. Treatment comprised methylprednisolone (dosage ranges from 3 g up to 18 g during disease course), cyclophosphamid or rituximab (with a minimum dosage of 2g), plasmapheresis or ivIG. We had no patients with a relapse in this cohort. None of the patients had a status epilepticus before lumbar puncture confounding the biomarker levels because of neuronal and axonal death due to the status epilepticus.

Two patients with other antibodies targeting extracellular epitopes [AMPAR and GABAB(R)] were not considered in the statistical analysis but for **Figure 3** to illustrate the biomarker and MRI timeline an AE patients. See **Table 1** for the different AE cohorts used per biomarker. We had follow up data in 13 patients. All other patients (samples) were categorized into either initial/acute phase or under treatment.

**Abbreviations:** Ab, Antibody; AD, Alzheimer's disease; AE, Autoimmune mediated Encephalitis; AMPAR, alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid receptor; Caspr2, Contactin associated protein 2; CSF, cerebrospinal fluid; FTD, Frontotemporal dementia; GABAB(R), Gabaaminobutyrat-B subunit receptor; Lgi1, Leucin-rich glioma inactivated protein1; NMDAR, N-methyl-D-aspartate receptor; NfL, Neurofilament light chain; PGRN, Progranulin; SE, Status Epilepticus; t-tau, T-tau; VGKC, Voltage-gated potassium channel.

#### TABLE 1 | Epidemiological data.

Overview of the single groups tested in this study. Number of patients included in bold, sex and mean age are listed.

#### Magdeburg Group

Patients with AE who were identified and treated in Magdeburg (n = 13) build a special cohort, because we could e.g., compare serial MRIs to look for AE caused lesions, basic CSF parameters, the outcome with the modified Rankin scale, t-tau, and other biomarker levels in the CSF and ab titer. The modified Rankin scale in this cohort was assessed by two experienced neurologist (PK, DB). Six out of thirteen had a paraneoplastic origin and one patient a postinfectious origin of the AE.

#### CSF-Neurofilament Light Chain Measurements

We divided the AE cohort into three groups regarding the CSF-NfL measurements in order to be able to perform a sufficient statistical analysis (see **Table 1**). One group with voltage-gated potassium channels (VGKC) mediated AE (comprising the Caspr2 and Lgi-1 patients) subdivided in an "initial" (n = 9) and "under treatment," meaning after several immunosuppressive therapies, subgroup (n = 8) and one group with NMDA patients under treatment (n = 13) (see **Table 1** and **Figure 1**). There were not enough NMDA patients who would fit into an initial/acute phase group (n = 3). Therefore, this group could unfortunately not be part of the statistical analysis.

Neurofilament light chain was measured with a commercial ELISA (Umandiagnostics, Sweden, catalogue number 10-7001 CE). The sensitivity of this assay is 31 pg/ml. The cut-off for pathological levels was set at 3523 ng/ml (mean (1823[ng/ml]) + 2 standard deviation (850[ng/ml]) above). Intraessay coefficient of variance is 7.4% and interessay coefficient of variance is 6%. NfL is a stable protein, which can be measured in the CSF even though the sample was on room temperature for up to 8 days (25). Therefore, we could measure NfL in samples not collected at Magdeburg, We used an already established control group at Magdeburg (n = 34, mean age = 64.4, CSF-NfL= 1823 ± 850 [ng/ml]) comprising patients with other than neuroinflammatory or neurodegenerative diseases (e.g., headache, suspicion of infection in the CNS etc.).

#### Total-Tau Measurements

The correct measurement of t-tau due to manufacturer's instructions requires different than standard processing of the CSF samples to create a cell-free sample excluding this parameter from a retrospective study. We yielded t-tau levels only in the Magdeburg group, as t-tau is part of the routine in Magdeburg but not in the other centers involved. Total-Tau levels were determined using a commercially available singleparameter ELISA kit [Innogenetics, Ghent, Belgium, catalogue numbers: 81572 (962-CE) and 81573] established in our routine diagnostical work up. Intraessay coefficient of variance is 13.2% and interessay coefficient of variance is 11.5%. The pathological levels were considered according to the manufacture guidelines.

#### CSF-Progranulin Measurements

We measured PGRN in CSF and serum of 36 Patients with AE. We divided our AE cohort (n = 36) into three groups according to the antibody causing the limbic encephalitis when looking statistically at the CSF-PGRN levels: one Lgi-1 group (n = 10, mean age = 69.2), one Caspr2 group (n = 8, mean age = 61.9) and a NMDAR group (n = 18, mean age = 27.1). These three groups were subdivided into two subgroups respectively one before and one after initiating immunosuppressive therapy (again, called "initial" or "under treatment") (see **Table 1** and **Figure 2**).

A commercial ELISA was performed to determine the levels of PGRN (Human Progranulin ELISA Kit, Mediagnost, Reutlingen, Germany, catalogue number E103) according to the manufacturer's instructions. Intraessay coefficient of variance is 4.4% and interessay coefficient of variance is 8.0%.

We established two control groups for PGRN measurements. Since PRGN levels are age dependent, we build a younger control group (n = 24; mean age 29.3 years; 18–40yrs) and one older group (n = 39; mean age 66.3 years,

50–75 years) and correlated it to age. The patients from these control groups had other neurological diseases than neuroinflammatory or neurodegenerative (e.g., acute headache, excluding neuroinflammatory diseases, no epilepsy patients). CSF-PGRN level was considered pathological, when the CSF-PGRN levels per control group were two standard deviation above or below the mean for each control group respectively.

interquartile range. White points mean outlier and star extreme outliers.

#### Antibody Detection

Antibody detection was either performed at the antibody laboratory Bielefeld, at the University Hospital Schleswig-Holstein, Department of Neuroimmunology Kiel and Lübeck or at the Institute of Molecular and Clinical Immunology Magdeburg. Standard indirect immunofluorescence tests were performed on antigen-specific transfected Hek293 cells as commercially available and used in clinical routine (EUROIMMUN, Lübeck, Germany, catalogue numbers: FA 112d-1003-6, FA 112d-1003-51, FA 1430-1003-1) for each patient revealing the specific antibody and the titer in CSF and serum.

#### Statistics

Statistics were calculated by SPSS 21.0 (IBM, Seattle, USA). Since group size and variances were not equal non-parametric tests were applied. For comparison of more than two different groups Kruskal-Wallis test was used with tamhanes post-hoc analysis. Group comparison of two groups was analyzed with Mann-Whitney-U test when they were independent and with the Wilcoxon test when the samples were paired to each other. For correlational analysis of serum and CSF PGRN Spearmann-rho correlation was applied. Tests were considered significant when reaching p < 0.05. There was no test for outliers applied.

### RESULTS

#### Cohorts

Since the CSF-PGRN level is age-dependent we established a younger control group aged 18–40 years (n = 24, CSF-PGRN= 0.72 ± 0.17 ng/ml, Serum-PGRN = 36.4 ng/ml, mean age = 29.3 years) and a control group 50-75 years (n = 39, CSF-PGRN = 0.94± 0.22 ng/ml, Serum-PGRN = 28.5 ng/ml mean age = 66.3 ± 9.8 years). Spearman correlation statistics revealed a significant correlation between CSF-PGRN and age (r = 0.275, p = 0.02).

In the Magdeburg group 9/13 had a FLAIR-intense signal in the limbic system on the MRI. Five out of thirteen patients developed a hippocampal sclerosis due to AE (see **Table 2** and **Figure 3**). Every patient with pathologically elevated t-tau levels developed a hippocampal sclerosis. The one patient, who developed a hippocampal sclerosis without elevated t-tau levels but elevated NfL concentrations, was administered after he was already treated and had a hippocampal sclerosis. Therefore the CSF was taken and measured ∼8 months after beginning of the AE and treatment. On the contrary only 4 out of 7 patients with pathological NfL levels developed a hippocampal sclerosis. Every marker of neurodegeneration and the modified Rankin scale (mRS) decreased after initializing the immunosuppressive treatment paralleled by a decrease in antibody titre (see **Figure 3** for examples and **Table 2** for the follow up data).

#### Neurofilament Light Chain

NfL was pathologically high (>3523 pg/ml) in 7/23 patients at different stages of the AE (see **Figures 2**, **3** and **Table 2**). Out of these seven patients, four had a paraneoplastic origin of the AE and one a postinfectious origin. Furthermore, 5/7 patients had a FLAIR-intense signal in the limbic areas on the MRI, which normalized during immunosuppressive treatment. This decrease in FLAIR signal was mirrored by a decrease in NfLlevels reaching normal NfL levels during disease course (see **Figure 3** and **Table 2**). Three out of five patients who developed hippocampal sclerosis had elevated CSF-NfL levels additionally to the also elevated t-tau. Solely elevated CSF-NfL was found in 4 patients. We correlated the leukocyte count to the NFL levels in the Magdeburg cohort and found no correlation (Spearmans r = 0.625).

There was a trend toward a lower NfL in the NMDA under treatment group (CSF-NfL = 1455 [pg/ml], range 142– 6841[pg/ml]) compared to the VGKC under treatment group (CSF-NfL = 2164 [pg/ml], range 821–4039 [pg/ml]) (Mann-Whitney U test p = 0.052 Z = −1.941), while there was no difference comparing the VGKC subgroups initial vs. under treatment (Wilcoxon test p = 0.735 and Z = −0.338).

#### Total Tau

Looking at the initial t-tau in our patients (before initiating the treatment) revealed a pathologically high t-tau in 4 patients (see **Table 2**). All 4 patients had MRI-FLAIR intense signals in the temporal lobe/limbic system and subsequently a hippocampal sclerosis. Immunosuppressive treatment did show an effect on TABLE 2 | Biomarkers in the Magdeburg cohort.

Complete list of all 13 patients in the cohort where follow up data is available including antibody, antibody serum titer, age, year at which the sample has been obtained, Neurofilament light chain (NfL), Total-tau (T-Tau), CSF-Progranulin (PGRN), Serum-Progranulin, cell count, modified Rankin scale (mRS) and comments on MRI abnormalities and putative pathogenesis; n.a., not available; #, timepoint before start of the immunosuppression.

the modified Rankin scale and also resulted in a decrease of ttau in these patients. The FLAIR signal also decreased in these patients (see **Figure 3**). In the other 8 patients without elevated t-tau levels immunosuppressive therapy had no effect on t-tau levels. A concomitant tumor had no impact at all on the t-tau levels. (see **Table 2**).

#### Progranulin

The mean CSF-PGRN levels were pathologically high in the initial NMDAR-group (CSF-PGRN=1.55 ± 1.1 ng/ml) reaching significance when compared to the NMDAR under treatment group (Mann-Whitney-U test Z = −2.5 and p = 0.012) and also when compared to the age-matched healthy group (Mann-Whitney-U test Z = −2.689 and p = 0.007). Serum PGRN levels were inside normal ranges in every AE patient and did not change after immunosuppression (see **Figure S1**). CSF-PGRN and CSF-Serum ratios ranged from 0.01 to 0.1, still CSF-PGRN is show to originate from the CNS when measured in the CSF (26, 27) We also could not find a correlation between Serum-PGRN and CSF-PGRN in all groups (Spearman-rho coefficient r = 0.17, p = 0.3) (**Figure S1** in the Supplement).After initiating the immunosuppressive therapy CSF-PGRN dropped to normal levels (CSF-PGRN = 0.75 ± 0.2 ng/ml) in the "under treatment" group.

Mean CSF-PGRN levels were normal in the Lgi-1 "initial" group (CSF-PGRN = 0.71 ± 0.11 ng/ml) and in the under treatment group (CSF-PGRN = 0.72 ± 0.12 ng/ml) (see **Figure 1**). Comparing these results to the age-matched group revealed significant differences (Kruskal-Wallis test: for "initial" PGRN: p = 0.009, X2 = 9.4, for follow-up: p = 0.04, X2 = 6.3). There were significantly lower levels in tamhane post-hoc in the Patients with AE (Lgi-1 initial vs. control p = 0.009 and p = 0.041 for the under treatment vs. control) The CSF-PGRN levels in both Caspr2 groups were inside the normal range (mean CSF-PRGN initially = 0.75 ± 0.23 ng/ml and mean CSF-PGRN under treatment = 0.8 ± 0.16 ng/ml) without significant results when compared to the age-matched controls (Caspr2 initial vs. under treatment p = 0.35 and Caspr2 under treatment vs. control p = 0.92). There was no difference between the "initial" and the "under treatment" group in the Lgi-1 and Caspr2 cohorts, respectively.

#### Follow Up Data

In summary, in all cases with an elevated biomarker of neurodegeneration in the CSF a decrease of biomarkers, ab titres, and mRS was observed following immunosuppressive treatment in all patients (see **Table 1** and **Figure 3**) regardless if the origin was postinfectious, paraneoplastic, or cryptogenic. There were two patients with all biomarkers simultaneously elevated. None of these parameters could predict a hippocampal sclerosis for sure on the one hand; on the other hand every patient who developed a sclerosis had either elevated t-tau or NfL levels with t-tau appearing to be more predictive.

### DISCUSSION

We could show for the first time that biomarkers of neurodegeneration originating from CNS are mirroring the clinical and probably neuroimmunological course of patients suffering from AE associated with antibodies to extracellular epitopes. CSF-PGRN is elevated in patients with NMDAR-AE during the acute phase. Furthermore, biomarkers of neurodegeneration such as t-tau together with CSF-NfL in Patients with AE might be predictive of the clinical outcome especially for developing a hippocampal sclerosis. The pathologically elevated biomarkers correlated with the mRS, the clinical course and the antibody titre. Besides Progranulin in acute NMDAR-AE, NfL, PGRN and t-tau did not seem to be restricted to one special autoantibody mediated AE. This may be due to the fact that the neuronal and axonal damages in general are mirrored and not the distinct pathomechanisms of each putative pathological autoantibody.

# Neurofilament Light Chain

NfL has been proven as an excellent marker of axonal loss (28). It seems very unlikely and there has been no data on whether peripheral tumors such as teratomas nor other neuroendocrine tumors can influence the CSF-NfL levels as possible confounders in our study. The co-occurrence of MRI changes, hippocampal sclerosis, and elevated NfL levels in our Magdeburg cohort is pointing at a pathomechanism causing the edema and subsequently the FLAIR signal resulting in the axonal dysfunction and subsequently increased NfL levels in the CSF. Other than in neurodegenerative diseases such as FTD (29, 30) the axonal loss in AE ceases after initiating sufficient immunosuppressive therapy as seen by the group of Pranzatelli in pediatric patients with opsoclonus-myoclonus syndrome caused by antibodies with intracellular epitopes (22, 23). Constantinescu et al. also measured CSF-NfL in four Patients with NMDAR-AE with AE and one Lgi-1 patient (19). Three NMDA-patients had a status epilepticus and highly pathological CSF-NFL levels, which has been seen in SE for nearly every marker of neuronal death (20, 21). In our cohort none had a status epilepticus confounding the biomarkers. This is possibly the reason why we found normal CSF-NfL levels in all measured Patients with NMDAR-AE except for the one with postinfectious origin. The meningoencephalitis with subsequent neuronal and axonal loss before the AE might be a reason for the elevated NfL levels in this patient since the infection was only 7 weeks apart from the AE. Our results are also much more in line with the known pathomechanism in NMDAR- AE (31, 32) where only marginal neuronal damage occurs and the main reason for the clinical symptoms is most likely the internalization of the NMDAreceptor. The one Lgi-1 patient (without SE) in the cohort of Constantiescu et al had normal NfL levels as our entire Lgi-1 group.

Total-Tau is a better marker for neuronal death as NfL (see below). However, FLAIR intense signals in the hippocampus as a consequence of disturbances of neuronal membrane function did correlate with NfL levels in our small cohort.

#### Total-Tau

Total-tau is an excellent marker for neuronal death (21, 33) 4/5 patients who developed a hippocampal sclerosis had pathological elevated t-tau levels in our Magdeburg cohort (n = 13). This is well in line with the current concept of the pathomechanisms leading to a sclerosis (34). Although, patient 1 (see **Table 2**) with the Caspr2 AE who was already treated months before admission to Magdeburg had only elevated CSF-NfL and normal t-tau levels and a hippocampal sclerosis (see **Table 2**). Sadly, it was not possible to measure NfL and t-tau levels in this patients initial CSF.

In sum, the measurement of t-tau may be a good marker before treatment decision in suspected autoimmune encephalitis or before deciding on the further immunosuppressive treatment but is limited to laboratories with expertise in measuring t-tau.

#### Progranulin

PGRN is playing a role in autoimmune mediated diseases such as rheuma or bowel disease or status epilepticus or in suppression of neuroinflammation (30, 35–37) Recently, EpiphrinA2 as a part of the Ephrin receptor kinase has been identified as functional receptor of PGRN and the potential of PGRN phosphorylating and activating the EpiphrinB2 receptor (38) linking it to the dysfunction in the EpiphrinB2 pathway known in AE mediated by autoantibodies against the NMDA-receptor (39, 40). The distribution in the fronto-temporal structures (41), the possible common link with the AE mediated by NMDAR via the EpiphrinA2-EpiphrinB2 pathway and the known role as a mediator in neuroinflammation and autoimmunity makes PGRN an interesting protein in AE.

We detected elevated CSF-PGRN levels in our NMDApatients with a severe ongoing AE. On the other hand, CSF-PGRN was low in patients suffering from Lgi-1-AE sometimes reaching levels of FTD patients (29) in contrast to Patients with NMDAR-AE and controls. The significance of this low CSF-PGRN is doubtful because none of the CSF-PGRN levels normalized after initiating the immunosuppressive therapy. Also most CSF-PGRN levels in the patients with Lgi-1-AE were still inside the normal range.

When looking at the t-cell or b-cell specific cytokine patterns in the patient's CSF suffering from NMDAR-AE several groups have seen a massive b-cell predominant cytokine pattern in the beginning especially in CXCL-13 levels (42, 43) and then a decrease in follow up. Also, cytokine pattern associated with t-cell activation were detectable throughout the course of AE without relevant changes. This course in cytokine levels could explain the elevated Progranulin levels in patients having acute NMDAR-AE.

PGRN in Serum and CSF was not elevated in patients with AE due to paraneoplastic origin although PGRN is also known as a tumor marker for certain tumors such as Lymphomas (44) The missing correlation between the serum-PGRN and CSF-PGRN is pointing at a cerebral origin of the CSF-PGRN as already seen in other diseases (26, 27). Overall, this result is probably due to an affection of the CSF-PGRN pathway in acute NMDAR-AE but needs more in vivo and in vitro experiments to be further examined.

One major limitation of the study is the small sample size in every subgroup tested. This fact is due to the very low numbers of patients with AE overall. Although total numbers are too small to draw a final conclusion the t-tau levels together with the CSF-NFL levels seem to best characterize the stage of neuronal death in the brain. The diagnostic value of NFL levels in the CSF should be evaluated in further studies. Another limitation is that we only had follow up data in 13 patients limiting our knowledge about MRI, mRS, and ab titres. Another limitation of the study is that due to the scarcity of the diseases measurements of the biomarkers could not be done in a batch but on demand.

A larger study should be conducted to further elucidate the correlation of these interesting parameters how they could contribute to therapeutic decisions.

#### CONCLUSION

NfL, t-tau and PGRN could be potential biomarkers of neuronal or axonal loss in patients suffering from AE. Especially, the Patients with NMDAR-AE have elevated PGRN levels at the acute phase of the AE. This fact further strengthens the hypothesis of a pathological change in the Epiphrin receptor metabolism in NMDAR patients. CSF-PGRN may be a marker for acute NMDA-AE.

Furthermore, we strongly recommend measuring NfL and ttau in the CSF of every patient with AE although one biomarker

#### REFERENCES

for itself could not predict all hippocampal sclerosis in this pilot study. Pathological levels of a biomarker of neurodegeneration should be considered as an on-going AE and may be taken into account when planning further therapy.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of ethics committee of the University hospital Magdeburg (number 100/16). The protocol was approved by the ethics committee of the University hospital Magdeburg. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

#### AUTHOR CONTRIBUTIONS

PK has access to all the data and takes responsibility for the data, accuracy of the data analysis, and the conduct of the research design or conceptualization of the study and analysis or interpretation of the data and drafting or revising the manuscript for intellectual content; HP and DB: Design and conceptualization of the study; analysis or interpretation of the data; drafting and revising the manuscript for intellectual content; LT: Conceptualization of the study; analysis of the data; drafting the manuscript for intellectual content; DV-W, JS-A, and SS: Conceptualization of the study; drafting the manuscript for intellectual content; WM, DR and FL: Conceptualization of the study; analysis and interpretation of the data; drafting and revising the manuscript for intellectual content; H-JH: Design of the study; drafting the manuscript for intellectual content.

#### ACKNOWLEDGMENTS

First, we have to thank our patients for the willingness to take part in this study. We have to thank Jeanette Witzke and Kerstin Kaiser at the neurochemical laboratory, Magdeburg for performing the excellent laboratory work. Furthermore, we have to thank Christian Bien at the Mara Epilepsy Center Bielefeld, Germany for laboratory work, samples and comments on the manuscript. We also appreciate the laboratory work of EUROIMMUN, Lübeck, Germany.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2018.00668/full#supplementary-material

control and prevention of cognitive impairment in a broadening phenotype. Brain (2013) 136:3151–62. doi: 10.1093/brain/awt212

antibodies: relation to antigenic specificity. J Neurol. (2014) 261:1695–705. doi: 10.1007/s00415-014-7408-6

**Conflict of Interest Statement:** PK has received consulting fees from Eisai (Germany). JS-A obtained honoraria for speaking engagements from Boehringer Ingelheim (Germany) and Bristol-Myers Squibb (Germany). LT: Saarland University, LT and others filed 61/730,772 which covers means and methods for detecting autoimmune disorders in which progranulin antibodies may be involved. FL runs an antibody detection laboratory in Kiel, Germany where part of the work has been performed.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Körtvelyessy, Prüss, Thurner, Maetzler, Vittore-Welliong, Schultze-Amberger, Heinze, Reinhold, Leypoldt, Schreiber and Bittner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

Boel De Paepe1 \*, Jana Zschüntzsch2 , Tea Šokcevi ˇ c´ 1 , Joachim Weis 3 , Jens Schmidt 2† and Jan L. De Bleecker 1†

1 Department of Neurology and Neuromuscular Reference Center, Ghent University Hospital, Ghent, Belgium, 2 Department of Neurology, University Medical Center Göttingen, Göttingen, Germany, 3 Institute for Neuropathology, Reinisch-Westfälische Technische Hochschule Aachen University Hospital, Aachen, Germany

We recently identified osmolyte accumulators as novel biomarkers for chronic skeletal muscle inflammation and weakness, but their precise involvement in inflammatory myopathies remains elusive. In the current study, we demonstrate in vitro that, in myoblasts and myotubes exposed to pro-inflammatory cytokines or increased salt concentration, mRNA levels of the osmolyte carriers SLC5A3, SLC6A6, SLC6A12, and AKR1B1 enzyme can be upregulated. Induction of SLC6A12 and AKR1B1 was confirmed at the protein level using immunofluorescence and Western blotting. Gene silencing by specific siRNAs revealed that these factors were vital for muscle cells under hyperosmotic conditions. Pro-inflammatory cytokines activated mitogen-activated protein kinases, nuclear factor κB as well as nuclear factor of activated T-cells 5 mRNA expression. In muscle biopsies from patients with polymyositis or sporadic inclusion body myositis, osmolyte pathway activation was observed in regenerating muscle fibers. In addition, the osmolyte carriers SLC5A3 and SLC6A12 localized to subsets of immune cells, most notably to the endomysial macrophages and T-cells. Collectively, this study unveiled that muscle cells respond to osmotic and inflammatory stress by osmolyte pathway activation, likely orchestrating general protection of the tissue. Moreover, pro-inflammatory properties are attributed to SLC5A3 and SLC6A12 in auto-aggressive macrophages and T-cells in inflamed skeletal muscle.

#### Edited by:

Tobias Ruck, Universität Münster, Germany

#### Reviewed by:

Tim Hagenacker, Universitätsklinikum Essen, Germany Corinna Preuße, Charité Universitätsmedizin Berlin, Germany

> \*Correspondence: Boel De Paepe<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology

Received: 20 August 2018 Accepted: 20 September 2018 Published: 11 October 2018

#### Citation:

De Paepe B, Zschüntzsch J, Šokcevi ˇ c T, Weis J, Schmidt J and ´ De Bleecker JL (2018) Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis. Front. Neurol. 9:846. doi: 10.3389/fneur.2018.00846 Keywords: inflammatory myopathy, osmotic stress, inflammatory stress, osmolytes, muscle regeneration

# INTRODUCTION

The idiopathic inflammatory myopathies represent a diverse group of autoimmune muscle diseases. The main disease entities recognized today are dermatomyositis (DM), polymyositis (PM), sporadic inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), anti-synthetase syndrome, and unspecific myositis, each of which possess distinct clinical and myopathological characteristics (1–5). DM patients develop complement-mediated blood vessel destruction, perimysial inflammation and perifascicular muscle fiber atrophy (6). PM and IBM are characterized by invasion of nonnecrotic muscle fibers by auto-aggressive cytotoxic T-cells and macrophages, with inflammation building up mostly at endomysial sites (7). In IBM muscle fibers, additional degenerative phenomena occur, with rimmed vacuoles and inclusions that contain aggregates of ectopic proteins (8), a process that is presumed to follow inflammation (9). IMNM represents 3 subdivisions according to serologic characteristics: anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase myopathy, signal recognition particle myopathy, and myositisspecific autoantibody negative IMNM (10). Many of the immunopathogenic mechanisms underlying the inflammatory myopathies remain poorly understood, hampering the development of successful therapies that suit the different patient subtypes.

Muscle is a highly adaptive and dynamic tissue capable of increasing its mass in response to exercise and of restoring damage caused by injury via processes that require hypertrophy and regeneration, respectively. In addition, cells possess a universal ability to adapt to changing osmotic conditions, a feature essential for their survival, allowing active anticipation toward perturbations in volume and disrupted cellular homeostasis. A complex intracellular mixture of interacting osmolytes regulates osmotic pressure, which takes shape through the synthesis and/or import of osmo-active compounds in cells (11). The solute carrier family (SLC) contains several saltdependent membrane transport proteins involved in the selective import of extracellular constituents: (i) the sodium-myoinositol cotransporter SLC5A3, (ii) the high-affinity taurine transporter SLC6A6, and (iii) the betaine and γ-amino-n-butyric acid (GABA) transporter SLC6A12. AKR1B1 is an aldose reductase that catalyzes the intracellular conversion of glucose to sorbitol. In a joint effort, these osmoprotective constituents represent a universal tool for mammalian cells, and their accumulators can be ubiquitously expressed throughout human tissues. The central regulator of osmolyte pathway gene expression is the transcription factor nuclear factor of activated T-cells 5 (NFAT5), also termed tonicity enhancer-binding protein (12).

Several observations allowed us to speculate NFAT5-inducible pathways might be involved in the inflammatory myopathies. Firstly, the NFAT5 pathway participates in muscle development and regeneration, by regulating the differentiation of immature myoblasts to mature multinucleated myotubes (13). NFAT5 levels have been shown to increase in the regenerating fibers of mice exposed to experimental muscle tissue injury (14), attributing the transcription factor a role in countering diseaseinflicted muscle tissue damage. Secondly, the NFAT5 pathway has been firmly linked to nuclear factor κB (NFκB) activity (15), the latter a key regulator of inflammatory diseases in general and the inflammatory myopathies in particular (16). NFAT5 and NFκB share multiple molecular targets (17), many of which are involved in the immunopathogeneses of inflammatory myopathy including CCL2, also termed monocyte chemoattractant protein-1 (MCP-1) (18), lymphotoxin β (LTβ) (19), tumor necrosis factor α (TNFα) (20), inducible nitric oxide synthase (iNOS) (21), and heat shock protein family of 70kd (HSP70) (22). Thirdly, NFAT5-downstream osmolyte pathways are potent activators of cytotoxic activities of immune cells and could therefore be implicated in human autoimmune disease. In addition, dietary salt is determinant to T-cell differentiation, by direct activation of glycogen synthase kinase 1 (GSK-1) and subsequent Interleukin 23 receptor stabilization, which enforces the type 17 helper T-cell (Th17), a T-cell phenotype associated with autoimmune disease (23).

We recently were the first to describe upregulation of the osmolyte accumulators SLC5A3, SLC6A6 and AKR1B1 in muscle tissues from myositis patients (24), and described NFAT5 expression in myoblasts in culture (25), yet the precise role osmolyte pathways play in disease mechanisms remained unexplored. With this study, we aim to substantiate and functionally connect these biomarkers with muscle inflammation. We set up in vitro muscle cell models to investigate osmolyte pathway member expression, using both the human rhabdomyosarcoma CCL-136 cell line and normal primary human myotubes. In addition, we investigated their possible signaling routes, which involves the upstream transcription factors NFκB and NFAT5, and the mitogen-activated protein kinases (MAPKs). We confronted this in vitro evidence with findings in muscle biopsies from patients diagnosed with inflammatory myopathies.

# METHODS

### Cell Cultures

Human rhabdomyosarcoma CCL-136 cells (ATCC, Manassas, VA) were kept in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany) and 1% L-glutamine (ThermoFisher Scientific, Waltham, MA). Human muscle cell cultures originated from muscle biopsies taken from healthy patients needing knee surgery, obtained with patient consent and approved by the local ethics committee. Biopsies were minced and trypsinized. Fragments were seeded in DMEM with pyruvate, high glucose and L-glutamine, supplemented with penicillin, streptomycin and 10% fetal calf serum (ThermoFisher Scientific). Cells were cultured for 2 periods of ∼ 3 weeks during which CD56+ cells were purified twice with magnetic separation MiniMACS columns using the supplier's standard protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Myotubes were obtained by allowing cells to differentiate for approximately 5 days in DMEM medium supplemented with penicillin, streptomycin and 2% horse serum (ThermoFisher Scientific). **Supplementary Figure S1** illustrates a representative culture of differentiated myotubes, showing aligned multinucleate muscle cells. All cells were kept in culture wells or chamber slides at 37◦C in a humified atmosphere containing 5% CO2. Conditions for cytokine stimulation were as determined earlier (26), being 30 ng/ml TNFα, 300 u/ml Interferon γ (IFNγ), 20 ng/ml Interleukin 1β (IL1β) (R and D Systems, Minneapolis, MN), or

**Abbreviations:** AKR1B1, aldose reductase**;** DM, dermatomyositis**;** dMyHC, developmental myosin heavy chain; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK-3α/β, glycogen synthase kinase 3α/β; HSP70, heat shock protein family of 70kd; IBM, sporadic inclusion body myositis; IFNγ, Interferon γ; IL1β, Interleukin 1β; IMNM, immune-mediated necrotizing myopathy; iNOS, inducible nitric oxide synthase; LTβ, lymphotoxin β; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemo-attractant protein-1; MSK2, mitogen- and stress-activated kinase 2; NFAT5, nuclear factor of activated T-cells 5; NFκB, nuclear factor κB; PM, polymyositis; siRNA, silencing RNA; SLC5A3, solute carrier sodium-myoinositol cotransporter; SLC6A6, solute carrier taurine transporter; SLC6A12, solute carrier betaine and γ-amino-n-butyric acid (GABA) transporter; Th17, Interleukin 17-producing T-cells; TNFα, tumor necrosis factor α.

double combinations. Hyperosmotic conditions were created by supplementing the culture medium with 25–150 mM of added NaCl. Higher concentrations of NaCl lead to complete cell death within 24 h.

# Quantitative Reverse Transcription PCR

RNA was prepared from cells cultured in 12-well culture plates, using the RNeasy Mini kit and according to the manufacturer's specifications (Qiagen, Hilden, Germany). RNA concentration was measured with a Nanodrop 1000 (ThermoFisher Scientific). cDNA was prepared from 200 ng of RNA with SuperScript II reverse transcriptase, 500 ng/µl oligo dTs, 0.1 M DTT, and 10 mM dNTPs each (Invitrogen, Darmstadt, Germany). cDNA was quantified through PCR reaction with Taqman Gene Expression Master mix (Applied Biosystems, Foster City, CA), using 6-carboxy-fluoresceinlabeled probes and specific primers for: SLC5A3, Hs00272857 \_s1; SLC6A6, Hs00161778\_m1; SLC6A12, HS00758246\_ m1; AKR1B1, Hs00739326\_m1; NFAT5, Hs00232437\_m1; MAPK14, Hs01051152\_m1; RELA, Hs00153294\_m1; NFKB1, Hs00765730\_m1; NFKB2, Hs01028901\_g1; glyceraldehyde-3 phosphate dehydrogenase (GAPDH), Hs99999905\_m1 (Applied Biosystems). Reactions were run in triplicate, following the standard cycle protocol on a 7500 Real Time PCR System, and analyzed with software version 2.0.6. (Applied Biosystems). Data was presented as 11Ct fold-changes compared to the expression levels in untreated cells, with GAPDH as an internal housekeeping gene standard.

### Immunofluorescent Cytostaining

Cells cultured on glass chamber slides were fixed with icecold acetone and blocked in phosphate buffered saline with 10% bovine serum albumin and 10% goat serum for 1 h at room temperature. Immunofluorescent detection was carried out for 1 h at room temperature with commercially available antibodies: mouse (4µg/ml, sc-514024, SantaCruz Biotechnology, Santa Cruz, CA) and rabbit (10µg/ml, nbp188641, Novus Biologicals, Abingdon, UK) anti-SLC6A12; rabbit (2µg/ml, sc-33219, SantaCruz Biotechnology) and goat (1µg/ml, sc-17732, SantaCruz Biotechnology) anti-AKR1B1; mouse anti-developmental myosin heavy chain (dMyHC) (40µg/ml, RMMy2/9D2, Leica Biosystems, Nussloch, Germany). The corresponding Alexa594—and Alexa488-labeled secondary antibodies (ThermoFisher Scientific) were added. After mounting in Fluoromount G (Southern Biotech, Alabama, USA), digital photography was performed on a Zeiss Axiophot microscope (Zeiss, Goettingen, Germany). Pictures were taken by a cooled CCD digital camera (Retiga 1300, Qimaging, Burnaby, BC, Canada) and visualized with ImageProPlus software (MediaCybernetics, Bethesda, MD).

#### Immunofluorescent Histostaining

For localization studies in muscle tissues, 8µm cryostat sections were cut from frozen muscle biopsies obtained from patients without muscle abnormalities (n = 10), inflammatory myopathies (n = 28), and disease controls diagnosed with muscular dystrophy (n = 8; for pathological information consult **Supplementary Table S1**). Diagnosis of the disease subgroups PM (n = 4), IBM (n = 9), DM (n = 9), and IMNM (n = 6) were based upon clinical and myopathological criteria (27). Diagnosis of PM was reserved to patients with nonnecrotic invaded muscle fibers present in the biopsy that had subsequently responded to immunosuppressive therapy. Sections were fixed in ice-cold acetone and treated with blocking solution containing 5% donkey serum, 10% heat-inactivated human serum and 2% bovine serum albumine in phosphate buffered saline. Incubations with primary antibodies were carried out in the same solution: 4µg/ml rabbit polyclonal anti-SLC5A3 (NBP1-02399, Novusbio); 4µg/ml mouse monoclonal anti-SLC6A6 (E10, SantaCruz Biotechnology); 6µg/ml rabbit polyclonal anti-SLC6A12 (HPA034973; Merck, Kenilworth, NJ); 1µg/ml goat polyclonal anti-AKR1B1 (N20, SantaCruz Biotechnology). Immune cell subtypes and muscle tissue constituents were visualized through double staining as described (18). Satellite cells were visualized with goat anti-Pax3/7 (1µg/ml, sc-7748, SantaCruz Biotechnology). To allow double staining of macrophages with mouse monoclonal antibodies, FITC labeled anti-CD68 (Agilent, Santa Clara, CA) was applied. Secondary antibodies were used labeled with CY3 (Jackson ImmunoResearch Laboratories, West Grove, PA) and AlexaFluor488 (ThermoFisher Scientific). Slides were mounted with Fluoromount (Southern Biotech) and visualized with a fluorescence microscope (Zeiss). Conventional semi-quantitative scoring of staining intensity was performed by three non-blinded independent observers. Negative control studies consisted of the omission of primary antibody and the substitution by non-immune IgGs. Positive control tissues for checking immunodetection were cultured Hela cells (SLC6A6), frozen sections containing kidney medulla (SLC5A3, SLC6A12), and Jurkat cells (AKR1B1).

#### Western Blotting

Cells cultured in 12-well culture plates were lysed in Ripa buffer (50 mM Tris-HCl, 150 mM NaCl, 2.5% Na-deoxycholate, 2.5% NP40, 0.1% sodium dodecyl sulfate pH 7.4) with a protease inhibitor cocktail added (Roche, Indianapolis, IN) and centrifuged for 5 min at 13000 rpm. The supernatant was collected and protein concentrations were determined following a Bradford procedure (Bio-Rad protein assay, Hercules, CA) with bovine serum albumine standard solutions, measured in triplicates on the Infinite M200Pro and analyzed with Magellan 7.2 software (Tecan, Mannedorf, Switzerland). 60 µg protein samples were dissolved in Laemli buffer, boiled for 2 min, separated by 12% sodium dodecyl sulfatepolyacrylamide gelelectrophoresis, and transferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Membranes were blocked with 5% bovine serum albumine for 1 h at 4◦C and incubated overnight at 4◦C with 2µg/ml mouse anti-SLC6A12 (sc-514024, SantaCruz Biotechnology), 4 h at room temperature with 2µg/ml goat anti-AKR1B1 (sc-17732, SantaCruz Biotechnology), and 1 h at room temperature with 0.7µg/ml mouse anti-GAPDH (Sp210-Ag14; Abcam, Cambridge, UK). All incubations were done in tris-buffered saline buffer containing 0.05% Tween20.

Appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were added for 1 h at room temperature. The chemiluminescent signal was generated with the Pierce Western blotting substrate (ThermoFisher Scientific), visualized with the Fusion FX, and quantified with Vision Capt software, with background noise filtering using a rolling-ball algorithm (Vilber Lourmat, Eberhardzell, Germany).

#### Protein Phosphorylation Profiling

Protein phosphorylation patterns were determined in extracts obtained from cultured myotubes, using the Proteome Profiler human phosphor-mitogen activated protein kinase (MAPK) antibody array according to the manufacturer's specifications (Bio-Techne, Abingdon, United Kingdom). Briefly, array membranes were incubated overnight at 4◦C with lysate containing 200 µg of cellular protein, detection conditions were as described in the western blotting section. Protein densities were quantified, relative to phosphorylated Akt2, as the calculated mean of duplicate spots per protein, using Image Studio 5.2 software (Li-Cor Biosciences, Cambridge, UK).

#### Knockdown Studies

Pools of three target-specific 19–25 nucleotide silencing RNAs (siRNAs), purchased from SantaCruz Biotechnology, were used: siRNA SLS5A3 (sc-44516), siRNA SLC6A12 (sc-95904), and siRNA AKR1B1 (sc-37119). 50% confluent CCL-136 cells and myotube cultures were changed to 500 µl X-Vivo15 medium (Sartorius, Goettingen, Germany), to which 3 µl of lipofectamine (ThermoFisher) and 100 nM siRNA had been added. After 5 h, an extra 200 µl of X-Vivo15 was added, which in treated cells contained cytokines to a final well concentration of 20 ng/ml IL1β+300 u/ml IFNγ, or 50 mM added NaCl. Cells were assayed 26 h after addition of the siRNAs. Live and dead cells were visualized using the ReadyProbes cell viability imaging blue/green kit (ThermoFisher) according to the manufacturer's specifications. Using a fluorescence microscope, a minimum of 50 cells (blue) was counted per condition, determining the amount of dead cells (green). Afterwards, cell cultures were stained with hematoxylin and eosin according to standard procedures, dehydrated, mounted, and interpreted under a light microscope. Efficiency of knockdown was evaluated at the mRNA level using quantitative reverse transcription PCR, following the method described above. From controls and siRNA-treated CCL-136 cells seeded in 12-well culture plates, protein samples were prepared for electrophoresis by adding lithium dodecyl sulfate sample buffer and reducing agent (Invitrogen, Carlsbad, CA, USA) and boiling for 3 min. Samples were loaded onto 10% bistris gels, with prestained markers alongside to determine the molecular weight of protein bands. Proteins were transferred to nitrocellulose membranes by electroblotting, and incubated with 2µg/ml mouse anti-SLC6A12 (sc-514024), 2µg/ml goat anti-AKR1B1 (sc-17732), or mouse anti-β actin (sc-47778) (SantaCruz Biotechnology) for 4 h at room temperature on a rocking platform. Immunoreaction was visualized using the chromogenic Western Breeze kit according to the manufacturer's specifications (Invitrogen).

# Compliance With Ethical Standards

Human experimentation presented in the study was approved by the Ghent University Hospital Ethics Committee (EC-UZG- #B670201316956) and adhered to privacy regulations (CBPL-BEL-#HM003039095). Written informed consent was obtained from all individual participants included in the study, and all procedrues were in accordance with the Declaration of Helsinki.

## RESULTS

#### mRNA Quantification in Cultured Muscle Cells

In CCL-136 cells, expression levels of osmolyte pathway members were determined in cells treated with pro-inflammatory cytokines or added NaCl for 24 h (**Table 1**). In general, a moderate increase of expression was shown for SLC5A3, SLC6A6, SLC6A12, and AKR1B1 when cells were treated with cytokines. The strongest response was a 10-fold increase of SLC5A3 mRNA expression in IL1β+TNFα-treated cells. Hyperosmotic conditions also induced osmolyte pathway expression, with 100 mM of added NaCl leading to a 179-fold increase of SLC6A12, and increasing NFAT5 expression 2.5-fold. MAPK14 levels were 2-fold increased in both IFNγ+TNFα– and 50 mM NaCl-treated cells. Highest levels of RelA, NFkB1 and NFkB2 could be achieved with pro-inflammatory cytokine mixtures, though NFkB1 and NFkB2 expression also responded to added NaCl.

Primary human myotubes treated for 24 h with cytokines or added NaCl showed induction of osmolyte pathway mRNA levels (**Table 2**). For SLC5A3 and AKR1B1, the strongest induction could be achieved with added NaCl; SLC6A6 and SLC6A12 were most strongly induced by pro-inflammatory cytokine mixtures. NFAT5 expression was influenced by both cytokines and increased salt concentrations, but reached higher expression levels with IFNγ+IL1β (4,5-fold) than with 125 mM NaCl (3 fold). MAPK14 expression levels were found increased 2-fold in myotubes treated with IL1β and with 125 mM NaCl. The expression of NFκB subunits was most strongly induced by treatment with pro-inflammatory cytokines. Nonetheless, 25 mM added NaCl increased NFkB1 expression 8,5-fold. In myotubes treated for longer periods, added NaCl lead to continuously increasing SLC5A3, SLC6A12 and AKR1B1 expression levels over time, culminating in levels all exceeding 200-fold at time point 72 h (**Table 3**). In addition, prolonged salt treatment lead to a time-dependent increase of NFAT5 and MAPK14 expression levels, reaching a maximum of 9-fold (NFAT5) and 18-fold (MAPK14). In contrast, the single pulse of pro-inflammatory cytokines resulted in highest expression levels after 24 h, with levels nearing normal after 72 h.

#### Immunofluorescent Protein Localization Studies in Cultured Muscle Cells

Immunofluorescent staining of CCL-136 cells (**Figure 1A**) confirmed the induction of SLC6A12 and AKR1B1 expression after 24 h of treatment with pro-inflammatory cytokines and

Fold changes compared to untreated cells, normalized to glyceraldehyde 3-phosphate dehydrogenase expression levels, were obtained through quantitative reverse transcription PCR and calculated as 2−11Ct (mean of three given). Fold changes >2 green; >5 yellow; >10 orange; >100 red.

TABLE 2 | Messenger RNA levels in cultured normal human myotubes treated for 24 h.

Fold changes compared to untreated cells, normalized to glyceraldehyde 3-phosphate dehydrogenase expression levels, were obtained through quantitative reverse transcription PCR and calculated as 2−11Ct (mean of three given). Fold changes >2 green; >5 yellow; >10 orange; >100 red.

added NaCl at the protein level and showed similar staining patterns with the two different sets of primary antibodies.

Myotubes immunostaining (**Figure 1B**) showed increases of SLC6A12 in response to cytokines at the 48 h time point, with levels declining again at time point 72 h. For AKR1B1, high levels were detected following cytokine stimulation from 24 h onward, compared to the low levels observed in untreated myotubes. A growth-inhibitory effect of hyperosmotic conditions was observed in myotubes, which became conspicuous from the 48 h time point on. NaCl-treatment disfavored differentiation into multinucleate elongated muscle cells, as was assayed with dMyHC staining (**Supplementary Figure S1**). Evaluation of hematoxylin&eosin stains confirmed that salt treatment affected cell morphology and growth, reducing cell elongation and disfavoring multinucleate myotubes.

# Protein Quantification and Phosphorylation Patterns of Human Myotubes

SLC6A12 protein levels were below the detection limit in untreated normal myotubes, but SLC6A12 protein could readily be shown in myotubes treated with IL1β+TNFα for 48 h and 72 h (**Figure 2A**). SLC6A12 and AKR1B1 protein levels were found to increase in a time- and dose-dependent manner when myotubes were exposed to 25 and 50 mM of added NaCl (**Figure 2B**). The expression levels with the higher doses of NaCl already reached a maximum at the 48 h time-point (**Table 4**).

Protein phosphorylation patterns of MAPKs showed that the strongest signals in the array were the phosphorylated forms of Akt2 (S474) and heat shock protein 27 (HSP27) (S78, S82) (**Supplementary Figure S2**). Akt2 and HSP27 activation was prominent in untreated and cytokine-treated myotubes alike. The most notable influence of 24 h IFNγ+IL1β treatment was an increase in phosphorylation of mitogen- and stress-activated kinase 2 (MSK2) (S360) 3-fold and MAPK3 (T202, Y204) 2.5 fold. In addition, levels of phosphorylated glycogen synthase kinase 3α/β (GSK-3α/β) (S9, S21), MAPK12 (T183, Y185), and MAPK13 (T180, Y182) were increased 2-fold.

#### Knockdown Studies in Cultured Muscle Cells

Both in CCL-136 cells and in primary healthy myotubes, 50 mM of added NaCl decreased cell densities in both siSCL6A12 and siAKR1B1-treated cells at the 24 h time point, while this regimen did not harm growth of siSLC5A3-treated muscle TABLE 3 | Messenger RNA levels in cultured normal human myotubes treated with cytokines or added NaCl for up to 3 days.

Fold changes compared to untreated cells, normalized to glyceraldehyde 3-phosphate dehydrogenase expression levels, were obtained through quantitative reverse transcription PCR and calculated as 2−11Ct (mean of three given). Fold changes >2 green; >5 yellow; >10 orange; >100 red.

cells (**Figure 3A**). Treatment with pro-inflammatory cytokines on the other hand, combined with knockdown of individual osmolyte pathway members, did not cause significant effects on myotube viability, with percentages of dead cells in untreated vs. cytokine-treated cells respectively: 1% vs. 7% (vehicle), 11% vs. 25% (siSLC6A12), 33% vs. 15% (siAKR1B1), and 0% vs. 4% (siSLC5A3). For silencing procedures, efficiency of knockdown was evaluated in CCL-136 cells (calculated as 1Ct siRNAtreated cells minus 1Ct of the vehicle control) detecting 16% (siSLC5A3), 59% (siSLC6A12), and 6% (siAKR1B1) of residual mRNA expression. Western blotting corroborated efficient AKR1B1 knockdown at the protein level but did not show a

substantial reduction of SLC6A12 protein levels (**Figure 3B**). We could not quantify SLC5A3 protein in muscle samples using western blots, as we were unsuccessful in detecting SLC5A3 in the corresponding positive control samples using rabbit (nbp102399, NovusBiologicals) and goat (sc-23142, SantaCruz Biotechnology) antibodies.

# Immunolocalization Studies in Muscle Tissues From Inflammatory Myopathy Patients

Confirming our earlier descriptive myopathological results on SLC5A3, SLC6A6, and AKR1B1 expression (24), we found these factors upregulated in regenerating muscle fibers present in biopsies in this new cohort of inflammatory myopathy patients. We now also add SLC6A12 to the regenerating muscle fiber's repertory of increased osmolyte accumulators (**Figure 4**). High osmolyte pathway member expression contrasted with the low levels present in muscle fibers in biopsies from healthy subjects. In tissues from muscular dystrophy patients, the SLC6A12 muscle fiber staining pattern was similar to inflammatory myopathies, with diffuse staining mostly in small regenerating CD56 positive muscle fibers and discontinuous sarcolemmal staining in subsets of CD56 negative muscle fibers (**Supplementary Figure S3**). We verified that this did not represent adjacent satellite cells and observed no co-localization with Pax3/7 staining.

TABLE 4 | Protein densities in cultured normal human myotubes treated with added NaCl for varying periods of time.

Protein densities of western blots (shown in Figure 2) were normalized using glyceraldehyde 3-phosphate dehydrogenase levels. Highest protein expression levels per time point have been highlighted.

SLC5A3 and SLC6A12 were expressed by inflammatory cells in inflammatory myopathy muscle biopsies (**Figure 4**). SLC6A12 was detected in subsets of the endomysial CD68+ cells surrounding muscle fibers in PM and IBM tissues. In comparison, only a small minority of tissue-infiltrating CD68+ cells in DM and in muscular dystrophy tissues were SLC6A12

positive, and the more sparse inflammatory cells observed in IMNM tissues were SLC6A12 negative. SLC6A12 was rarely observed in the CD8+ T-cells of PM and IBM tissues. SLC5A3 on the other hand, could be detected in a substantial part of CD3+ T-cells, with most non-invading CD8+ T-cells being negative while CD8+ cells actively invading nonnecrotic muscle fibers in PM and IBM were often strongly positive. The majority of CD68+ cells, and part of the CD206+ cells were also SLC5A3 positive. In contrast, inflammatory cells were invariably AKR1B1 and SLC6A6 negative in all muscle tissues tested.

# DISCUSSION

Our earlier studies had identified osmolyte pathway members as biomarkers for inflammatory myopathies, yet the pathogenic routes behind their elevated expression remained unknown. The present study offers further context and allows us to speculate on the underlying mechanisms.

# Inflammatory Stress Induces Osmolyte Pathways in Muscle Cells

We here report that, in addition to increased NaCl concentrations, pro-inflammatory cytokines can induce osmolyte pathways in muscle cells in an in vitro setting. In response to osmotic stress, normal primary myotubes displayed a continuous increase of SLC5A3, SLC6A12, and AKR1B1 mRNA expression levels over time, while single cytokine pulse led to highest mRNA levels after 24 h, steadily decreasing afterwards and returning to near-normal levels at the 72 h time point. Immunocytochemical staining and western blotting experiments confirmed the transient induction pattern of SLC6A12 at the protein level. We cannot rule out fast cytokine degradation in the culture medium, yet this is unlikely as many have

reported prolonged effects by single pulse cytokine treatments over several days (28–30). Using silencing techniques, we showed that compromising osmolyte accumulator expression negatively influenced cell growth only when muscle cells faced hyperosmotic conditions. We produced strongest evidence for AKR1B1, for which the knockdown regimen severely reduced AKR1B1 protein levels, yet did not compromise growth when myoblasts and myotubes were challenged with pro-inflammatory cytokines. Based upon this data, we speculate on different levels of importance of the osmolyte pathway dependent upon the challenges muscle cells face: osmolytes being essential protectors against osmotic stress while having a less vital yet regulatory role in the response to inflammatory stress. Our observations would need to be confirmed and analyzed further with knockdown combinations, as an important additional facet is the degree of redundancy of function displayed by the different osmolyte pathway members. In evidence, only SLC5A3 deficiency represents a lethal murine phenotype (31), with knockout of AKR1B1, SLC6A6, and SLC6A12 resulting in viable mice suffering from limited defects in renal function (32–34). The compensatory role of remaining osmolytes when activity of single pathway members is compromised, has long been confirmed in kidney cells (35).

Transcription factors NFκB and NFAT5 represent key regulators of cell's responses to inflammatory stress on the one hand and osmotic stress on the other hand. Not surprisingly therefore, pro-inflammatory cytokines were the most potent inducers of RelA, NFkB1, NFkB2 expression in muscle cells. However, we did observe some effects of NaCl-treatment on NFκB expression levels and, vice versa, NFAT5 expression responded to pro-inflammatory cytokines. Thus, muscle cells' responses to inflammatory and osmotic stress appear not separately regulated via NFκB and NFAT5, respectively.

The MAP kinases, a large family of Ser/Thr kinases that translate cell surface signals to the nucleus, play a crucial role in inflammation (36), which led us to study their involvement in muscle cells' responses to pro-inflammatory cytokines. We found Akt2 and HSP27 were most heavily phosphorylated in myotube cultures, but their activation was unaltered in response to inflammatory cytokines. Akt signaling influences muscle development and regeneration, affecting either initiation (Akt1) or maturation (Akt2) of myotubes (37). HSP27 is also associated with myotube differentiation and was found absent from myoblasts (38). The strong activation of Akt2 and HSP27 we observed in our in vitro model fits with the differentiated stage of the myotubes we studied in our experiments. The MAPKs of the p38 subgroup have been put forward as complex regulators of osmolyte pathways, through their hypertonicity-induced phosphorylation, and have been implicated in inflammatory disease (39). Our study found MAPK14 mRNA expression increased up to 2-fold by pro-inflammatory cytokines, and we observe increased phosphorylation of MAPK12 and MAPK13 in human primary myotubes treated for 24 h with IL1β+IFNγ. Of the extracellular signal-regulated kinases (ERK) on the other hand, we observed increased phosphorylation of MAPK3 (ERK1) in IL-1β+IFN-γ-treated myotubes. This is in line with our recent study that showed cytokines induce ERK1/2 phosphorylation, which subsequently leads to protein deposition and autophagy in muscle cell cultures (40). We propose that the 24 h response of muscle cells to pro-inflammatory cytokines may thus be regulated primarily via such phosphorylation-driven activation of residential MAPKs. Muscle cells exposed to osmotic stress for prolonged periods appear to require extra measures for their protection, culminating in 18-fold (MAPK14) and 9-fold (NFAT5) increases in expression levels.

# Osmolyte Pathways Are Activated in Regenerating Muscle Cells

Osmolytes are known regulators of skeletal muscle development, with the involvement of taurine and betaine already described in most detail. Taurine is essential for skeletal muscle buildup, and knockout mice lacking its transporter SLC6A6 display severe structural defects (41) and exercise intolerance (42). The trimethylglycine betaine appears also important for proper muscle functioning, although SLC6A12 knockout mice have been reported to develop only mild myopathy (34). Betaine promotes muscle fiber differentiation and myotube size (43) via stimulation of the mechanistic target of rapamycin pathway (44) and disturbed osmolyte balances have been implicated in muscle disease. Taurine levels were significantly reduced in muscle from myositis patients compared to healthy controls (45), while in urine on the other hand both taurine and betaine levels were increased in patients (46). In the murine Duchenne muscular dystrophy model, taurine content of muscle was low, mostly early in disease progression, and a reduction of its transporter SLC6A6 was observed (47). Yet, in the canine golden retriever muscular dystrophy model, muscle levels of taurine and SLC6A6 were 1.5- and 20-fold increased (48) compared to healthy dogs, pointing to possible differences between species and/or at different disease stages. Our localization studies revealed strong increases of osmolyte accumulators in small regenerating fibers. Possibly, protein replacement and refolding during the regeneration process are chaperoned by osmolytes, the latter aiding protection of functional protein conformations (49).

# Subsets of Muscle-Infiltrating Inflammatory Cells Express SLC5A3 and SLC6A12

In addition to the muscle cells' general and dynamic repertory of osmolyte accumulators, we found two osmolyte pathway members, SLC5A3 and SLC6A12, selectively expressed in subsets of macrophages and T-cells infiltrating skeletal muscle. Interestingly, we observed an association with active invasion of nonnecrotic muscle fibers, a phenomenon typically observed in PM and IBM muscle tissues (50), which fits with current notions of how osmolytes behave as potent cytotoxic regulators. Cytotoxic macrophages have been shown to accumulate betaine, myoinositol and taurine as compatible organic osmolytes in response to osmotic stress, via MAPK-regulated upregulation of osmolyte transporters (51, 52). It is well known that macrophages have versatile functionalities, with a spectrum stretching out between the classical inflammatory M1 phenotype, to the alternative anti-inflammatory, tissue repair-oriented M2 phenotype (53). Significantly higher levels of AKR1B1 mRNA and protein have been reported in M1 macrophages compared with M2-polarized macrophages (54). The upstream transcription factor NFAT5 is enhanced by the M1-promoting pro-inflammatory and hypoxic conditions associated with autoimmune diseases, which is particularly suggested to regulate the chemokine MCP-1 and subsequent synovial macrophage survival in rheumatoid arthritis (55). We found a subset of CD68+ cells, regarded as representing mostly M1 macrophages, to express SLC5A3 and SLC6A12, most prominently in PM and IBM tissues. In addition to macrophages, T-cells have also been shown to engage NFAT5-regulated pathways in their development and activation (56) and, in accordance, we found SLC5A3 and SLC6A12 expression in a minority of T-cells. In helper T-cells (Th-cells), salt-induced NFAT5 activity promotes their differentiation into Interleukin 17-producing Th-cells (Th17-cells) (57). This is relevant to inflammatory myopathy, as IL-17 induces and maintains chronic inflammation, and Th17 cells represent a pathogenic subset of T-cells associated with inflammatory myopathy (58).

# CONCLUSIONS

The expression of osmolyte pathway members extends to human tissues that normally are not exposed to hypertonicity, which has led to the assumption that these factors have additional more versatile functions. In this respect, they have been put forward as biomarkers for inflammation (59) and tumor metastasis (60). The data we presented here points to a general role for osmolyte accumulation, via AKR1B1, SLC5A3, SLC6A6, and SLC6A12 upregulation, in muscle cells challenged by inflammatory stress, presumably in an attempt to stabilize protein function in sight of the changed proteome during regeneration. In addition, our data suggest an individual inflammatory role for SLC5A3 and SLC6A12 as potential regulators of the myocytotoxicity displayed