Research articleDown-regulation of purinergic P2X7 receptor expression and intracellular calcium dysregulation in peripheral blood mononuclear cells of patients with amyotrophic lateral sclerosis
Introduction
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, primarily characterised by degeneration of upper and lower motor neurons (MNs) resulting in generalised muscular weakness and paralysis. Disease onset usually appears between the ages of 40 and 70 years. The median survival rate is 3–5 years following symptoms onset, with respiratory failure being the most frequent cause of death [1].
The pathophysiological mechanisms that contribute to MN cell death include mitochondrial dysfunction, oxidative stress, excitotoxicity, immune factors and apoptosis. A critical feature of MN death is the dysregulation of intracellular Ca2+ concentration [Ca2+]i [2]. The endoplasmic reticulum (ER) interacts with adjacent structures and plays an important role in Ca2+ signalling [3]. Ca2+ circulates between the ER and mitochondria; described as the ER–mitochondria Ca2+ cycle (ERMCC) [4]. These mechanisms are essential for normal function of MNs; however, they might also cause the demise of MNs under pathological conditions [5], [6]. The function and survival of MNs is also dependent on the environment comprised of non-neuronal cells including astrocytes, microglia, T lymphocytes and monocytes.
ALS is regarded as a multi-systemic disorder. Typical changes of the disease can be observed in peripheral blood mononuclear cells (PBMCs) [7]. Abnormally activated monocytes were found in blood specimens from patients with sporadic ALS, suggesting that these cells play a role in the pathogenesis of ALS and could serve as biomarkers [7], [8]. Furthermore, increased activation of immune cells was detected in spinal cords and surrounding tissues from ALS patients [9]. Regarding PBMCs, disturbances in purinergic signalling may promote neurodegeneration [10] and the expression of P2X receptors (P2XR) are of special interest [11]. P2XRs are involved in neuronal processes and their activation by adenosine triphosphate (ATP) leads to elevation of intracellular Ca2+ and cellular depolarisation. Specifically, P2X4R and P2X7R are key receptors in neurodegenerative diseases [12], [13].
Various studies report that immune cells, including PBMCs, affect the survival of MNs [14], however detailed mechanisms underlying the activation of the peripheral purinergic signalling in ALS remain unclear.
This study was performed to investigate the expression of purinergic P2X4 and P2X7 receptors and to determine putative Ca2+ dysregulation in PBMCs via activation of P2X4 R and P2X7 R by ATP.
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Subjects and blood samples
The study was performed at the Department of Neurology at the Jena University Hospital and including 42 ALS patients diagnosed according to the revised El Escorial criteria [15] and 19 healthy controls. Disease severity was assessed by the revised ALS Functional Rating Scale (ALSFRS-R). Disease progression was calculated as (48-ALSFRS-R)/disease duration. ALS patients received Riluzole but no psychotherapeutic drugs. 18 ml of blood was drawn into vacuum EDTA tubes. The study was approved by the
Patients
The mean age of the ALS patients (27 male and 15 female subjects) was 61.8 ± 11.7 years. The mean ALSFRS-R score was 36.7 ± 8.1 from a maximum score of 48 in healthy individuals. The mean disease duration was 21.8 ± 13.0 months and the mean progression rate was 0.75 ± 0.64. 26 patients had spinal onset, whereas 16 had bulbar onset. The control group consisted of 10 females and 9 males with a mean age of 58.6 ± 12.0 years.
Downregulation of P2X7R expression in monocytes
Typical PBMC morphologies were observed using light microscopy (Fig. 1A). The
Discussion
ALS is a multisystemic disease in which pathological processes extend beyond MNs. Specifically, PBMCs display traits of the disease including changes in immunophenotype [17], increased oxidative stress [18] and glutamatergic dysfunction [19]. PBMCs could serve as a promising biomarker based on the low invasiveness and high availability of material.
The vulnerability of MNs and intracellular Ca2+ disturbances are known features in ALS and have been studied in ALS animal models. MNs are
Conclusion
The study confirms that Ca2+ abnormalities in ALS do not only exist in motor neurons but also in immune cells. Purinergic signalling events regulate the activation and functional responses of PBMCs. Our study improves the understanding of ALS pathophysiology on a systemic level and suggests that PBMCs could serve as non-invasive surrogate markers in ALS. To confirm the direct link between reduced P2X7R expression and Ca2+ disturbances in PBMCs of ALS patients functional studies are required.
Acknowledgements
We thank Svetlana Tausch for technical assistance and Mandy Arnold for blood collection. We express thanks to Nasim Kroegel for language editing. This study was undertaken in cooperation with the German MND-NET. The project is supported by the German Bundesministerium für Bildung und Forschung (BMBF) grant SOPHIA to JG under the aegis of the EU Joint Programme − Neurodegenerative Disease Research (JPND − www.jpnd.eu) and a BMBF grant PYRAMID to JG in the framework of the ERANET E-RARE program (//www.e-rare.eu
References (34)
- et al.
Role of mitochondria in kainate-induced fast Ca2+ transients in cultured spinal motor neurons
Cell Calcium.
(2007) - et al.
Calcium, a signaling molecule in the endoplasmic reticulum?
Trends Biochem. Sci.
(2000) - et al.
Calcium dysregulation in amyotrophic lateral sclerosis
Cell Calcium.
(2010) - et al.
Calcium dysregulation, mitochondrial pathology and protein aggregation in a culture model of amyotrophic lateral sclerosis: mechanistic relationship and differential sensitivity to intervention
Neurobiol. Dis.
(2011) - et al.
ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis
Exp. Neurol.
(1999) - et al.
Evidence for systemic immune system alterations in sporadic amyotrophic lateral sclerosis (sALS)
J. Neuroimmunol.
(2005) - et al.
Extracellular A.T.P. and P.2X7 receptors in neurodegeneration
Eur. J. Pharmacol.
(2002) - et al.
MCP-1 chemokine receptor CCR2 is decreased on circulating monocytes in sporadic amyotrophic lateral sclerosis (sALS)
J. Neuroimmunol.
(2006) - et al.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
(1985) - et al.
Immune system alterations in sporadic amyotrophic lateral sclerosis patients suggest an ongoing neuroinflammatory process
J. Neuroimmunol.
(2009)
Peroxiredoxin I. is a ROS/p38 MAPK-dependent inducible antioxidant that regulates NF-kappaB-mediated iNOS induction and microglial activation
J. Neuroimmunol.
P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ
Cell Calcium
Genetic background effects on disease onset and lifespan of the mutant dynactin p150Glued mouse model of motor neuron disease
PLoS One
Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells
PLoS One
Inflammation in amyotrophic lateral sclerosis spinal cord and brain is mediated by activated macrophages, mast cells and T cells
Amyotroph Lateral Sc.
Extracellular A. T. P. and neurodegeneration
Curr. Drug Targets CNS Neurol. disord.
Transcript analysis of P2X receptors in PBMCs of chronic HCV patients: an insight into antiviral treatment response and HCV-Induced pathogenesis
Viral Immunol.
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