Gene Expression Analysis of Astrocyte and Microglia Endocannabinoid Signaling during Autoimmune Demyelination

Abstract

The endocannabinoid system is associated with protective effects in multiple sclerosis (MS) that involve attenuated innate immune cell responses. Astrocytes and microglia are modulated by endocannabinoids and participate in the biosynthesis and metabolism of these compounds. However, the role of neuroglial cells as targets and mediators of endocannabinoid signaling in MS is poorly understood. Here we used a microfluidic RT-qPCR screen to assess changes in the expression of the main endocannabinoid signaling genes in astrocytes and microglia purified from female mice during the time-course of experimental autoimmune encephalomyelitis (EAE). We show that astrocytes and microglia upregulate the expression of genes encoding neurotoxic A1 and pro-inflammatory molecules at the acute disease with many of these transcripts remaining elevated during the recovery phase. Both cell populations exhibited an early onset decrease in the gene expression levels of 2-arachidonoylglycerol (2-AG) hydrolytic enzymes that persisted during EAE progression as well as cell-type-specific changes in the transcript levels for genes encoding cannabinoid receptors and molecules involved in anandamide (AEA) signaling. Our results demonstrate that astrocytes and microglia responses to autoimmune demyelination involve alterations in the expression of multiple endocannabinoid signaling-associated genes and suggest that this system may regulate the induction of neurotoxic and pro-inflammatory transcriptional programs in both cell types during MS.

Mesenchymal stem cells instruct a beneficial phenotype in reactive astrocytes

“…Thus, in parallel with the in vitro data showing that MSC attenuate the RNA expression of the A1 marker Serpin G1 that was induced by exposure of astrocytes to IFN‐γ, we immunolocalized complement factor C3, as marker of A1 phenotype in vivo, on brain sections from naïve and EAE‐affected mice treated or not with MSC. Our data confirmed a previous report (Moreno‐García et al, 2020) indicating that reactive astrocytes in EAE overexpress the A1 marker C3, and demonstrated that administration of MSC diminished the number of C3‐expressing A1 astrocytes to the levels detected in naïve mice (Figure 3a). Thus, in order to establish whether or not MSC, besides attenuating the A1 phenotype, may promote the acquisition of a neuroprotective trait in reactive astrocytes, we immunolocalized S100A10 as a marker of A2 astrocytes, in the same experimental groups.…”

Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects

“…We now demonstrate that microglia CB2R mRNA level was 162 ± 63-fold higher than that of neurons in adult mouse brain. Since CB2R in microglia is also inducible [ 57 , 58 ], the purified microglia CB2R expression may not reflect that of naïve microglia. Our finding of increased expression of neuron CB2R by methamphetamine treatment agrees with previous findings of neuron CB2R upregulation by brain stressors such as cocaine [ 59 ], traumatic brain injury [ 60 ], seizure [ 61 ], and ischemia [ 62 ].…”

Neurological Benefits, Clinical Challenges, and Neuropathologic Promise of Medical Marijuana: A Systematic Review of Cannabinoid Effects in Multiple Sclerosis and Experimental Models of Demyelination

“…In EAE, increased endocannabinoid levels in the brain, cerebellum, and plasma of C57BL/6 mice treated with a selective endocannabinoid reuptake inhibitor, WOBE437, correlated with significant muscle relaxation, decreased neuroinflammatory infiltration, and lower microglial proliferation [187]. Dynamic transcriptional deregulation in the endocannabinoid system during EAE results in an early shift toward enhanced 2-AGmediated CB1 receptor activation in astrocytes that is followed by deficits at later stages of the disease [188].…”