Ablation of P2X7 receptor exacerbates gliosis and motoneuron death in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motoneurons. The primary triggers for motoneuron degeneration are still unknown, but inflammation is considered an important contributing factor. P2X7 receptor is a key player in microglia response to toxic insults and was previously shown to increase pro-inflammatory actions of SOD1-G93A ALS microglia. We therefore hypothesized that lack of P2X7 receptor could modify disease features in the SOD1-G93A mice. Hetero- and homozygous P2X7 receptor knock-out SOD1-G93A mice were thus generated and analysed for body weight, disease onset and progression (by behavioural scores, grip and rotarod tests) and survival. Although the lifespan of P2X7(+/-) and P2X7(-/-)/SOD1-G93A female mice was extended by 6-7% with respect to SOD1-G93A mice, to our surprise the clinical onset was significantly anticipated and the disease progression worsened in both male and female P2X7(-/-)/SOD1-G93A mice. Consistently, we found increased astrogliosis, microgliosis, motoneuron loss, induction of the pro-inflammatory markers NOX2 and iNOS and activation of the MAPKs pathway in the lumbar spinal cord of end-stage P2X7(-/-)/SOD1-G93A mice. These results show that the constitutive deletion of P2X7 receptor aggravates the ALS pathogenesis, suggesting that the receptor might have beneficial effects in at least definite stages of the disease. This study unravels a complex dual role of P2X7 receptor in ALS and strengthens the importance of a successful time window of therapeutic intervention in contrasting the pathology.

Clemastine Confers Neuroprotection and Induces an Anti-Inflammatory Phenotype in SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

“…In this context, we also provided evidence that while eliciting beneficial effects, clemastine downregulated P2X7 in lumbar spinal cord, thus validating the concept that purinergic P2X7 receptor is a player in the pathogenesis of ALS in SOD1 G93A mice [21,40]. This is consistent with our previous results showing that inhibition of P2X7 during a well-defined phase of ALS disease exerts neuroprotection and reduces neuroinflammation [40].…”

“…Transgenic progeny was genotyped analyzing tissue extracts from tail tips, according to the following procedure. Briefly, tail tips were digested (overnight, 55°C) in tail buffer (100 mM Tris-HCl, pH 8, 0.5 % Tween 20, 0.5 % NP40) plus 200 mg/ml proteinase K, and then heated at 75°C for 20 min to inactivate proteinase K. The presence of SOD1 G93A mutant transgene was assessed by performing PCR on digested tails, by using BioMix Red (Bioline) and the following primers: SOD1 forward 5′-CATCAGCCCTAATCCATCTGA-3′; SOD1 reverse 5′-CGCGACTAACAATCAAAGTGA-3′ [21].…”

“…In order to assess mice general condition, starting at 6 weeks of age, we adopted a behavioral score system based on a scale from 1 to 5 as follows: 5= healthy without any symptom of paralysis; 4=slight signs of destabilized gait and paralysis of the hind limbs; 3=obvious paralysis and destabilized gait; 2=fully developed paralysis of the hind limbs, animals only crawl on the forelimbs; and 1= fully developed paralysis of the hind limbs, animals predominantly lie on the side, and/or are not able to straighten up within 30 s, after turning them on the back [21]. After reaching a score of 1, the animals were euthanized, according to the guidelines for preclinical testing and colony management [23].…”

Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery occlusion

“…It is thus tempting to speculate that the protective M2 phenotype may critically rely on an intact P2X7. Consequently, a lacking functional P2X7 receptor in M2 microglial cells may cause the detrimental effects observed 24 h after MCAO in the absence of a functional P2X7 receptor, as has been proposed before [36]. On the other hand, there is extensive evidence that antagonism of P2X7 exerts neuroprotective effects, presumably by preventing intracellular Ca 2+ overloads.…”

Role and Therapeutic Potential of Astrocytes in Amyotrophic Lateral Sclerosis

“…The purinergic receptor P2X7 is a key player in microglial response and ATP released from astrocytes may increase pro-inflammatory actions of ALS microglia through activation of this receptor [163, 164]. However, ablation of P2X7 receptor exacerbates gliosis and motor neuron death in hSOD1 G93A mice [165]. These results may be explained by the observation that astrocyte-mediated deactivation of microglia may be detrimental in ALS models.…”