Identification of a Biomarker in Cerebrospinal Fluid for Neuronopathic Forms of Gaucher Disease

March Identification of a Biomarker in Cerebrospinal Fluid for Neuronopathic Forms of Gaucher Disease Hila Zigdon 0 1 2 Alon Savidor 0 1 2 Yishai Levin 0 1 2 Anna Meshcheriakova 0 1 2 Raphael Schiffmann 0 1 2 Anthony H. Futerman 0 1 2 0 1 Department of Biological Chemistry, Weizmann Institute of Science , Rehovot , Israel , 2 de Botton Institute for Protein Profiling, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science , Rehovot , Israel , 3 Institute of Metabolic Disease, Baylor Research Institute , Dallas, TX , United States of America 1 Data Availability Statement: The authors have included all individual data points in an attached Excel file, and the proteomics data has been submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001654 , username: 2 Academic Editor: Stephen D Ginsberg, Nathan Kline Institute and New York University School of Medicine, UNITED STATES Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available. - Gaucher disease (GD), the most common lysosomal storage disease (LSD), is caused by mutations in the GBA1 gene, which encodes for glucosylceramidase (GlcCerase), the lysosomal hydrolase responsible for glucosylceramide (GlcCer) degradation [1]. GD is classically divided into three clinical sub-types based on age of onset and on signs of nervous system involvement [2]. Type 1 is the chronic, non-neuronopathic form and types 2 and 3 are the acute and chronic neuronopathic forms, respectively, which display central nervous system (CNS) involvement in addition to systemic disease [3], and are collectively known as neuronopathic GD (nGD). However, the disease encompasses a wide spectrum of phenotypes and a great diversity in severity and symptoms is observed in patients classified as the same sub-type. Thus, the Competing Interests: The authors have declared that no competing interests exist. manifestation of disease can be described as a phenotypic continuum. An effective treatment, enzyme replacement therapy, is available for type 1 GD but no therapies are available for nGD, although attempts are being made to identify possible therapeutic targets [4,5]. However, because of the wide heterogeneity of symptoms displayed by nGD patients, the efficacy of candidate drugs would be immensely facilitated by the availability of genuine biochemical biomarkers. Moreover, interest in GD and nGD has recently been boosted by the realization that heterozygous mutations in GBA1 are a major risk factor for Parkinsons disease [6], leading to the suggestion that GD therapies might be of use for treating Parkinsons disease [7]. In the current study, we performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantitative proteomics to identify biochemical markers in the cerebrospinal fluid (CSF) of four type 3 GD patients and five controls, and identified a protein, glycoprotein non-metastatic B (GPNMB), whose levels in the CSF reflect diseases severity. This was confirmed in a series of studies in which GD was induced in mice and GPNMB levels monitored in the CSF. We suggest that GPNMB can be used as an authentic biochemical marker to follow the progression of nGD pathology and the efficacy of potential treatments. Materials and Methods Human brain and CSF samples The spinal fluid samples were collected for biomarker discovery from a clinical trial a phase I/ II randomized, controlled study of OGT 918 in patients with neuronopathic GD (Clinicaltrials.gov identifier NCT00041535) [8]. The samples were collected under a study that was overseen by the Institutional Review Board (IRB) of the National Institute of Neurological Disorders and Stroke (NINDS), National Institute of Health (NIH). All patients or their legal guardians gave their written informed consent for their participation. Following a waiver of consent received from NINDS IRB, these samples became part of the Repository Protocol Institute of Metabolic Disease that is overseen by the IRB of Baylor Research Institute, Dallas, Texas. The stated purpose of this study was to To support the neurometabolic research using in human samples and data in the Institute for Metabolic Diseases, Baylor Research Institute, Baylor University Medical Center. Samples were anonymised prior to shipment. All patients were on long-term enzyme replacement therapy (ERT) as well as on Miglustat; note that neither have any therapeutic effect on the brain [8] All patients eye movement abnormalities [9] and had not undergone splenectomy. Human brains were provided by the University of Miami Brain and Tissue Bank for Developmental Disorders through NICHD contract NO1-HD-8 3284 [10]. All control brains were frozen within 626 h of death. GD patients were classified before death as types 1, 2 or 3 based on the clinical course of the disease, and in most cases, mutational analysis was also performed. Brains from GD patients were obtained post-mortem with informed consent between 7 and 22 h after death. After removal, brains were frozen on dry ice. Mice were maintained under specific pathogen-free conditions and handled according to protocols approved by the Weizmann Institute Animal Care Committee according to international guidelines. Gbaflox/flox; nestin-Cre mice were used as a model of nGD, in which GlcCerase deficiency is restricted to neurons and macroglia [11,12]. nGD was also induced in C57BL/ 6OlaHsd mice by intra-peritoneal injection with 100 mg/kg/day conduritol B-epoxide (CBE) (Calbiochem), an irreversible GlCerase inhibitor [13]. Proteins were reduced by incubation with 5 mM dithiothreitol (Sigma-Aldrich) for 30 min at 60C followed by alkylation with 10 mM iodoacetamide (Sigma-Aldrich) in the dark for 30 min at 21C. Proteins were subsequently digested with trypsin (Promega) overnight for 6 h followed by trypsin for 16 h at 37C. Digestions were stopped by additi (...truncated)