Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis - PubMed (original) (raw)

Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

Savina Apolloni et al. Dis Model Mech. 2014 Sep.

Abstract

In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a 'gene modifier' in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by interfering with the neuroinflammatory component of the disease.

Keywords: ALS; Brilliant Blue G; Microglia; Motor neuron; P2X7.

© 2014. Published by The Company of Biologists Ltd.

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Figures

Fig. 1.

Fig. 1.

Treatment with BBG starting at 100 days delays onset and ameliorates disease progression in SOD1-G93A mice. (A) Behavioural scores were improved upon treatment using the BBG100 protocol (red, _n_=16), with respect to vehicle-treated mice (black, _n_=34), whereas there were no differences using the BBG70 (green, _n_=8) and BBG40 (blue, _n_=14) protocols. (B) The disease onset in SOD1-G93A mice was at 20 weeks, as determined by a decrease in rotarod performance at 15 r.p.m. The disease onset of BBG100-treated mice was delayed to 21 weeks. (C) SOD1-G93A mice showed a decline in rotarod performance with respect to WT mice (brown, _n_=6), but those treated using the BBG100 protocol showed an improvement in rotarod performance with respect to vehicle-treated mice. (D) All BBG-treated mice showed no differences in median survival with respect to vehicle-treated SOD1-G93A mice, as shown by Kaplan–Meier survival curves (158 days for the BBG100 protocol, 161 days for the BBG70 protocol and 161 days for the BBG40 protocol vs 163 days for mice treated with the vehicle). Each group of transgenic mice was statistically evaluated with respect to its proper control (vehicle group at each time point, _n_=10–12). In accordance with historical data of our colony, the behavioural, rotarod and survival curves of vehicle-treated mice were overlapping, regardless of the starting week of treatment, and thus cumulated to simplify the graphical representation. *_P_≤0.05.

Fig. 2.

Fig. 2.

Microgliosis but not astrocytosis is decreased in BBG-treated SOD1-G93A mice. (A) Equal amounts of total lumbar spinal cord lysates from WT mice (6 months) and SOD1-G93A mice treated with vehicle or the BBG100 and BBG40 protocols at end stage (_n_=4) were subjected to western blotting using Iba-1 and β-actin for protein normalization. The panel on the right shows quantification of the blots relative to WT mice. (B) Spinal cord sections (L3-L5) from WT mice (6 months) and SOD1-G93A mice that had been treated with vehicle or the BBG100 protocol at end stage were stained using an antibody against Iba-1. BBG100-treated SOD1-G93A sections showed a decrease in Iba-1 labelling with respect to those from vehicle-treated SOD1-G93A mice (_n_=4–5 per group). (C) Spinal cord sections (L3-L5) from WT mice (6 months) and SOD1-G93A mice treated with vehicle or the BBG100 and BBG40 protocols at end stage were stained for CD68. (D) Quantitative analysis of CD68 in the ventral horns of spinal cord shows that CD68 optical density increases in vehicle-treated mice (dark grey) with respect to WT mice (white bar) and significantly decreases only upon treatment using the BBG100 protocol (grey bar), but not the BBG40 protocol (black bar), when compared with vehicle (_n_=4–5 per group). (E) Spinal cord sections (L3-L5) from WT mice and end-stage SOD1-G93A mice that had been treated with vehicle or the BBG100 and BBG40 protocols were stained for GFAP. Data represent means±s.e.m.; Student’s _t_-test compared with WT, *P<0.05; or with vehicle-treated SOD1-G93A, #P<0.05. Scale bars: 100 μm.

Fig. 3.

Fig. 3.

BBG inhibits the expression of NF-κB and modulates M1 and M2 markers in SOD1-G93A mice at end stage. (A) Equal amounts of lumbar spinal cord lysates from WT mice (6 months) and SOD1-G93A mice treated with vehicle or the BBG100 and BBG40 protocols at end stage (_n_=3) were subjected to western blotting and immunoreactions for NF-κB p65. β-actin was used for protein normalization. The chart on the right shows quantification of the blots relative to WT mice. RNA was extracted from lumbar spinal cords of WT mice (6 months) and SOD1-G93A mice treated with vehicle or the BBG100 and BBG40 protocols at end stage (_n_=4), and the expression profiles of (B) the M1 markers NOX2 (left-hand panel) and IL-1β (right-hand panel) and (C) the M2 markers BDNF and IL-10 were examined by using qRT-PCR. WT, white bars; vehicle, dark grey bars; BBG100, grey bars; BBG40, black bars. Data represent means±s.e.m.; Student’s _t_-test compared with WT, *P<0.05; or with vehicle-treated SOD1-G93A mice, #P<0.05.

Fig. 4.

Fig. 4.

BBG influences NOX2 expression in SOD1-G93A mice. (A) Equal amounts of lumbar spinal cord lysates from WT mice (6 months) and SOD1-G93A end-stage mice treated with vehicle or the BBG100 and BBG40 protocols (_n_=3) were subjected to western blotting and immunoreactions for gp91phox (also known as NOX2). β-actin was used for protein normalization. The chart on the right shows quantification of the blots relative to WT mice. (B) Spinal cord sections (L3-L5) from WT (6 months), vehicle, BBG100 and BBG40 SOD1-G93A mice at end stage were immunostained for gp91phox (green). In the merged insets (yellow), the presence of gp91phox is shown in microglial cells stained for Iba-1 (red). (C) BBG-treated mice showed a decrease in gp91phox optical density, as compared with vehicle-treated SOD1-G93A mice (_n_=4–5 per group). WT, white bars; vehicle, dark grey bars; BBG100, grey bars; BBG40, black bars. Data represent means±s.e.m.; Student’s _t_-test compared with WT, *P<0.05; or with vehicle-treated SOD1-G93A mice, #P<0.05. Scale bars: 50 μm (B); 25 μm (inset of B).

Fig. 5.

Fig. 5.

Decreased motor neuron loss in SOD1-G93A mice treated with BBG starting at 100 days. (A) Spinal cord sections (L3-L5) from WT mice (6 months) and SOD1-G93A mice treated with vehicle or the BBG100 and BBG40 protocols at end stage were stained with Cresyl Violet to assess motor neuron numbers. SOD1-G93A mice display an evident loss of Nissl substance with respect to WT, and this effect is attenuated in mice treated using the BBG100 protocol (B). Quantitative analysis of motor neurons in the ventral horns of spinal cord shows that vehicle- (dark grey) and BBG40-treated (black bar) SOD1-G93A mice exhibit a similar reduction of motor neuron number (35% and 37%, respectively) when compared with WT sections (white bar). BBG100-treated mice show a ~35% increase in motor neurons as compared with vehicle-treated SOD1-G93A mice (_n_=4–5 per group) (Student’s _t_-test compared with WT, #P<0.05; or with vehicle-treated SOD1-G93A, *P<0.05). (C) Motor neurons of spinal cord sections (L3-L5) from WT mice and SOD1-G93A mice that had been treated with vehicle or the BBG100 protocol at end stage are stained with anti-ChAT. Scale bars: 100 μm (A), 50 μm (C).

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