Astroglia acquires a toxic neuroinflammatory role in response to the cerebrospinal fluid from amyotrophic lateral sclerosis patients

Mishra et al. Journal of Neuroinflammation (2016) 13:212 DOI 10.1186/s12974-016-0698-0 RESEARCH Open Access Astroglia acquires a toxic neuroinflammatory role in response to the cerebrospinal fluid from amyotrophic lateral sclerosis patients Pooja-Shree Mishra1,2, Dinesh K. Dhull1,3, A. Nalini4, K. Vijayalakshmi1, T. N. Sathyaprabha1, Phalguni Anand Alladi1 and Trichur R. Raju1* Abstract Background: Non-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF). Methods: Various inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, or Student’s t test, as applicable. Results: Here, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells. Conclusions: Our study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS. Keywords: Neuroinflammation, Astrocytes, Cytokines, ROS, COX-2, Trophic factors, ALS Abbreviations: NC, Normal control; NALS, Non-amyotrophic lateral sclerosis/non-degenerative neurological diseases; ALS, Amyotrophic lateral sclerosis; IL-6, Interleukin-6; TNF, Tissue necrosis factor; IFN-γ, Interferon gamma; IL-10, Interleukin-10; NO, Nitric oxide; iNOS, Inducible nitric oxide synthase; ROS, Reactive oxygen species; PGE2, Prostaglandin E2; COX-2, Cyclo-oxygenase 2; VEGF, Vascular endothelial growth factor; GDNF, Glial cell linederived neurotrophic factor; NC-ACM, Normal control astroglia conditioned medium; ALS-ACM, ALS-CSF exposed astroglia conditioned medium; NALS-ACM, NALS-CSF exposed astroglia conditioned medium; NSC34, Neuroblastoma X spinal cord hybrid cell line; ANOVA, Analysis of variance * Correspondence: 1 Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore 560029, India Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mishra et al. Journal of Neuroinflammation (2016) 13:212 Background Etiopathogenesis of amyotrophic lateral sclerosis (ALS), the devastating and relentlessly progressing neurodegenerative disorder leading to muscular weakness, is poorly understood. Approximately 90 % of the total cases reported have unknown etiology and are categorized as sporadic ALS. However, the rest 10 % of the cases follow an autosomal dominant inheritance pattern (familial ALS/FALS), and only ~20 % of these may be mapped to the mutations in the SOD-1 gene, which forms the basis of the animal models of ALS [1]. Apart from the occurrence of neuronal death, reports from these mutant SOD-1 transgenic models, as well as the autopsy studies, have demonstrated the non-cell autonomous contribution of the astrocytes in ALS [2–4]. The activated astrocytes may adopt either a neuroprotective or a neurotoxic phenotype in a stimulusdependent manner by a process termed as glial polarization; the end-results of which depend largely on the microenvironment experienced by the astrocytes [5, 6]. For instance, a neuroprotective role of astrocytes has been thoroughly discussed in various pathological conditions including stroke and spinal cord injury [7]. On the other hand, astrocytes also respond in a toxic manner in response to excess ATP or the inflammatory factors like interleukin (IL)-1β and free radicals released by M1 microglia [8]. Apart from the increased expression of GFAP and S100β, activated astrocytes may respond by regulating certain inflammatory, trophic, and/ or toxic factors in the milieu that may act directly on neurons or through other immune cells. These include pro/ anti-inflammatory cytokines, inflammatory markers, and trophic factors [9, 10]. The plausible role of astrocytes in the initiation/progression of ALS has been studied employing chimeric mSOD1/TDP-43 models or human iPSC-derived astrocytes from ALS patients [11–13]. Such models have elucidated the possible toxic role of astrocytes in the pathophysiology of ALS. Lepore et al. [14] investigated the effect of transplantation of astroglial precursor cells into the spinal cord of mSOD-1 mice to establish healthy astroglial pools and demonstrated the mitigation of the disease symptoms, including a reduction in microgliosis. These findings suggest the important role of astrocytes in modulating the inflammatory response. Glutamate-associated excitotoxicity following the selective loss of astroglial glutamate transporters, leading to reduced synaptic glutamate uptake, has been reported in the autopsy samples as well as the animal models of ALS [3, 15]. However, along with the reduction in glutamate uptake, possibility of pathological release of glutamate by astrocytes as the source of the abnormal elevation in the glutamate levels in ALS should also be considered. Some of the early work done indeed suggests Page 2 of 14 excessive glutamate release in experimental models of familial ALS, but the source remains unknown [16, 17]. 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