Significance of elevated Prohibitin 1 levels in Multiple Sclerosis patients lymphocytes towards the assessment of subclinical disease activity and its role in the central nervous system pathology of disease

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Abstract

Multiple Sclerosis (MS) is an autoimmune-neurodegenerative disorder managed therapeutically by modulating lymphocytes activity which has potential in disease management. Prohibitin 1(PHB) that controls the reactive oxygen species (ROS) and present on the activated lymphocytes have significance in the therapy of MS as esters of fumaric acid that regulates ROS is in phase II/III clinical trials. Thus, we evaluated the expression levels of PHB1 in experimental autoimmune encephalomyelitis (EAE), the animal model of MS and on MS patient’s lymphocytes. PHB levels in brain tissue of EAE animals were determined by immunoblotting and on blood lymphocytes from MS relapse, Remission, Optic Neuritis, Neurological controls and Healthy volunteers by FACS using anti-PHB and anti-CD45 antibodies. We observed significant elevation of PHB in EAE brains (91.0 ± 17.59%) vs controls (29.8 ± 12.9%) (p = 0.01) and on lymphocytes of MS patients in acute (73.5 ± 11.20%) or relapsing (69.3 ± 17.33%) phase compared to remission (45.9 ± 8.08%) [p = 0.034 acute vs remission; p = 0.004 relapse vs remission]. Up regulation of PHB in relapsing vs remission MS patients imply the potential use of PHB to clinically evaluate subclinical disease status towards prognosis of an oncoming relapse. Elevated PHB levels in EAE brains signify the role of PHB in regulating ROS and implies PHB’s role in oxidative stress.

Introduction

MS is an autoimmune disease in which activated lymphocytes against self-antigens of the CNS contributes immensely to the development of the disease [[1], [2]]. At present MS is managed therapeutically to alleviate subclinical disease activity leading to reduced symptoms [[3], [4]]. Of much importance to clinicians would be the ability to predict when a patient may imminently move again to active disease state, or to determine whether a particular treatment is leading the patient towards remission using a simple blood test. MRI evaluation of lesions has not been a sensitive or successful indicator of such prognostic activity as changes in lesions only occur at the onset of visible and significant demyelination [[5], [6]]. What has been typically observed is that there is a flare up in autoimmune activity in terms of activated lymphocytes and other proinflammatory responses which precede neuronal damage and demyelination [7]. Although the up regulated cytokines, chemokine, neurofilaments, fetuin A, myelin basic protein as well as osteopontins from CSF have been suggested as contenders for subclinical disease activity for MS [[8], [9], [10], [11]], withdrawing CSF every time is clinically not practical. The alternative may be to monitor these responses in the peripheral blood; however, inflammatory cytokines in the peripheral blood is not always related to subclinical disease activity of MS and may not measure lymphocyte activity alone [12].

In this study we have selected Prohibitin-1 (PHB) as a potential molecular marker for analyzing subclinical disease activity in the peripheral blood due its over expression found in activated T cells [13]. PHB protein is ubiquitously expressed and found to be located in the mitochondria, nucleus and plasma membrane. It has been found that the PHB is associated with anti-proliferative activity, hence the name prohibitin. To this reason, PHB is also upregulated in cancers may be to prevent its proliferation. Indeed, prohibitin execute its effect in different cell types by inhibiting cell proliferation [[14], [15], [16]]. PHB mRNA injection blocks DNA synthesis, and stimulation of DNA synthesis were observed with antisense oligonucleotide [17]. It has been shown that prohibitin antibodies down regulated the CD3 induced signaling which normally lead to T cell proliferation [13]. Moreover, neurological injuries, PHB overexpression has been shown and it protect neurons from reducing free radical production by mitochondria [18]. Thus, we began with the premise that PHB could be a specific marker during remission and active phase of MS. We sought to measure PHB levels in peripheral blood lymphocytes in patients under remission and under the active phase of the disease and compared the same results with other neurological controls. We also quantified PHB levels in the brains of animals suffering from experimental autoimmune encephalomyelytis (EAE) which is an animal model of MS. The results are then discussed in terms of the utility of PHB as a prognostic marker for active and remitting phases of the disease. We recognize that previous studies have shown a correlation of PHB with activated lymphocytes as shown in leukemia [[19], [14], [13]], but not in MS patients. This will be the first study to explore PHB as a potential biomarker in MS patients to distinguish between relapse and remitting phases of the disease.

Section snippets

Patient demographic details

Blood samples were collected from healthy volunteers and patients as per the guidelines approved by the institutional ethics committee after informed consent from patients. All samples were obtained from patients attending MS clinic of Neurology Department, at Amrita Institute of Medical Sciences, Cochin, Kerala, India. We enrolled patients with clinically definite MS comprising acute phase MS, relapsing MS and MS under remission according to revised McDonald’s criteria [20] (Table 1, and

PHB is elevated in PBMC of MS patients

We analyzed initially the expression of PHB in PBMC of relapsing MS patients and compared it with healthy volunteers (Table 1, Table 2section 1) in order to detect changes in PHB expression levels. As shown in Fig. 1, significantly increased expression of PHB (343.3 ± 105.6) is seen in the PBMC’s compared to control healthy individuals (27.4 ±19.0) (p=0.039). Although, the MS sample #2 is an outlier based on the results seen. However, this sample was used for quantification because both actin

Discussion

PHB is an evolutionary conserved ubiquitously expressed protein localized to multiple cellular compartments such as mitochondria, nucleus, and the plasma membrane [26]. Most importantly, increased expressions of prohibitins on the cell surface were induced remarkably upon T cell activation were also reported [[13], [11]]. PHB is involved in the negative regulation of T cell receptor signalling leading to reduced T cell proliferation and that PHB deficiency led to inflammation indicating PHB’s

Conflict of interest

None

Acknowledgements

This work is supported by a grant from Indian Council of Medical Research (ICMR), IRIS ID 2011-09420 to Dr Krishnakumar Menon. Mr SMK is supported by the Council of Scientific and Industrial Research (CSIR) senior research fellowship. We also acknowledge gratefully the support given by the Center for Nanosciences and Molecular Medicine for the infrastructure.

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