Upregulation of CB2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis.

María Fernández-Trapero,Carmen Rodríguez-Cueto,Javier Fernández-Ruiz,Francisco Espejo-Porras,Joan R Coates,Carmen Pérez-Díaz,Eva De Lago

doi:10.1242/dmm.028373

Abstract

ABSTRACTTargeting of the CB2 receptor results in neuroprotection in the SOD1G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors are facilitated by their upregulation in the spinal cord of the mutant mice. Here, we investigated whether similar CB2 receptor upregulation, as well as parallel changes in other endocannabinoid elements, is evident in the spinal cord of dogs with degenerative myelopathy (DM), caused by mutations in the superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem spinal cords from unaffected and DM-affected dogs. Tissues were used first to confirm the loss of motor neurons using Nissl staining, which was accompanied by glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated possible differences in the expression of endocannabinoid genes measured by qPCR between DM-affected and control dogs. We found no changes in expression of the CB1 receptor (confirmed with CB1 receptor immunostaining) or NAPE-PLD, DAGL, FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly elevated in DM-affected dogs determined by qPCR and western blotting, which was confirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling colocalized with GFAP but not Iba-1, indicating upregulation of CB2 receptors on astrocytes in DM-affected dogs. Our results demonstrate a marked upregulation of CB2 receptors in the spinal cord in canine DM, which is concentrated in activated astrocytes. Such receptors could be used as a potential target to enhance the neuroprotective effects exerted by these glial cells.

Highlights

Amyotrophic lateral sclerosis (ALS) is a progressive degeneration and loss of upper and lower motor neurons in the brain and spinal cord, causing muscle weakness and paralysis (Hardiman et al, 2011)
Such elevation of cannabinoid receptor type-2 (CB2) receptors has been described in SOD1G93A transgenic mice (Shoemaker et al, 2007; MorenoMartet et al, 2014), and we recently found that the response occurred predominantly in activated astrocytes recruited at lesion sites in the spinal cord (F.E.P., unpublished results)
We found that CB2 receptor immunolabelling colocalized with GFAP immunofluorescence (Fig. 6), indicating that the upregulation of CB2 receptors in the spinal cord of degenerative myelopathy (DM)-affected dogs occurred in reactive astrocytes

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive degeneration and loss of upper and lower motor neurons in the brain and spinal cord, causing muscle weakness and paralysis (Hardiman et al, 2011). Similar studies have identified mutations in other genes, such as TARDBP (TAR-DNA binding protein) and FUS (fused in sarcoma), which encode proteins involved in pre-mRNA splicing, transport and/or stability (Buratti and Baralle, 2010; LagierTourenne et al, 2010) and, in particular, the CCGGGG hexanucleotide expansion in the C9orf gene, which appears to account for up to 40% of genetic cases (Cruts et al, 2013) Their pathogenic mechanisms, which differ, in part, from the toxicity associated with mutations in SOD1, led to a novel molecular classification of ALS subtypes (Al-Chalabi and Hardiman, 2013; Renton et al, 2014). Based on Disease Models & Mechanisms (2017) 10, 551-558 doi:10.1242/dmm.028373 these studies, the CB2 receptor may be a novel target in altering disease progression in ALS, given its effective control of glial influences exerted on neurons, as investigated in other disorders (Fernández-Ruiz et al, 2007, 2015; Iannotti et al, 2016 for review)

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Conclusion