Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis

Varghese et al. Clinical Proteomics 2013, 10:19 http://www.clinicalproteomicsjournal.com/content/10/1/19 CLINICAL PROTEOMICS RESEARCH Open Access Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis Anu Mary Varghese1†, Aparna Sharma1†, Poojashree Mishra1, Kalyan Vijayalakshmi1, Hindalahalli Chandregowda Harsha2, Talakad N Sathyaprabha1, Srinivas MM Bharath3, Atchayaram Nalini4, Phalguni Anand Alladi1 and Trichur R Raju1* Abstract Background: Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease. Results: Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia. Conclusion: Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS. Keywords: Proteomics, Cerebrospinal fluid, Sporadic amyotrophic lateral sclerosis Background Selective loss of cortical and spinal motor neurons is the characteristic feature of Amyotrophic Lateral Sclerosis (ALS), an adult onset progressive fatal neurodegenerative disorder. Factors predisposing the most prominent form of this multifactorial disease viz. sporadic ALS (SALS) remain obscure due to the difficulties in developing animal models. Therefore, development of novel therapeutics is also severely hampered. This is also largely attributed to the lack of a ‘biomarker’ which can be objectively measured as an indicator of pathogenic processes and/or pharmacologic response to therapeutic interventions [1]. The discovery of ideal biomarkers may offer tools for rapid diagnosis, monitoring disease * Correspondence: † Equal contributors 1 Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Hosur Road, Post Box no.: 2900, Bangalore 560 029, India Full list of author information is available at the end of the article progression and provide insights into the pathophysiology of the disease; thereby broadening therapeutic options. Proximity to the central nervous system (CNS) renders Cerebrospinal Fluid (CSF) to be the ideal biofluid for detection of biomarkers in CNS pathologies. It is speculated that toxic agents which propagate the disease are synthesized in the affected areas, injure the neighboring cells, and are released into the extracellular space and CSF [2,3]. We have earlier shown that exposure of embryonic rat spinal cord cultures to ALS-CSF (in-vitro) and intrathecal injection of the same into neonatal rats (in-vivo) induced degenerative changes in motor neurons and showed the involvement of astrocytes [4-11]. Intracerebroventricular infusion of ALS-CSF in adult rats perturbed the cortical motor neuronal activity and was associated with poor motor performance [12]. Thus several studies, including ours, support the presence of © 2013 Varghese et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Varghese et al. Clinical Proteomics 2013, 10:19 http://www.clinicalproteomicsjournal.com/content/10/1/19 toxic factor(s) in ALS-CSF and attribute them a role in eliciting its pathophysiology [3,13-15]. We undertook a study to identify the toxic factor(s) in ALS-CSF through proteomic analysis. Quantitative mass spectrometric analysis of ALS-CSF compared to agematched controls showed upregulation of 31 proteins, amongst which Chitotriosidase-1 (CHIT-1) showed more than 10 fold increase. The biological significance of CHIT1 expression is intriguing in view of the absence of its natural substrate chitin in human brain. Earlier studies have shown an increase in CSF CHIT-1 levels in multiple sclerosis (MS) and Alzheimer’s disease (AD) [16,17]. Page 2 of 9 Results Confirmation of toxicity of ALS-CSF samples The toxicity of the ALS-CSF samples was confirmed prior to proteomic analysis. Exposure of NSC-34 cells to the CSF obtained from ALS patients (ALS-CSF) resulted in a dramatic decrease in their viability when compared to the control groups; i.e. the cells treated with CSF from normal individuals (N-CSF) or without CSF (NC) (***p < 0.001 vs NC; ###p < 0.001 vs N-CSF; Figure 1A). It also induced enhancement of LDH activity in ALS-CSF group (***p < 0.001 vs. NC and $$$p < 0.001 vs. N-CSF; Figure 1B). Figure 1 Mass spectrometric analysis of ALS-CSF samples: Toxicity assays (A & B) were performed. Histogram of MTT assay showing 40% reduction in the viability of NSC-34 cells upon exposure to 10%(v/v) ALS-CSF compared to the cells exposed to normal-CSF (###p < 0.001 vs. N-CSF) and 30% compared to normal controls (***p < 0.001 vs. NC; A). ALS-CSF caused significant increase in LDH activity when compared to control groups (***p < 0.001 vs. NC and $$$p < 0.001 vs. N-CSF; B). Gel image representing depletion of abundant proteins prior to mass spectrometric analysis (C). Representative MS/MS spectra of the peptides of CHIT1, Osteopontin, CHI3L1 and CHI3L2 (D). Tests of significance was Student’s t test, One way Anova followed by Tukey’s post hoc analysis. Varghese et al. Clinical Proteomics 2013, 10:19 http://www.clinicalproteomicsjournal.com/content/10/1/19 Page 3 of 9 Proteomic analysis of control and ALS-CSF samples Validation of LC-MS/MS data by enzyme assay Ten CSF samples each from controls and ALS were pooled, depleted of abundant proteins and electrophoresed on SDS-PAGE (Figure 1C). Total protein from control and ALS-CSF were subjected to tryptic digestion, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling with isobaric tags for relative and absolute quantitation (iTRAQ). LCMS/MS analysis identified 819 proteins using SEQUEST and Mascot (Additional file (...truncated)