Analysis of endocannabinoid receptors and enzymes in the post-mortem motor cortex and spinal cord of amyotrophic lateral sclerosis patients

Abstract

Objective: We have investigated the endocannabinoid system in the motor cortex of motor neuron disease (MND) patients. Methods: Post-mortem samples from MND patients and controls were used for immunostaining and/or Western blotting analysis of endocannabinoid elements. Results: We did not find any evidence of neuronal losses in the motor cortex of MND patients, but elevations in glial markers Iba-1 and GFAP were evident. We found no changes in FAAH and MAGL enzymes and in the CB₁ receptor, which correlated with the lack of cortical neuron death. By contrast, the Western blotting analysis of CB₂ receptors proved an increase in the motor cortex corroborated by immunostaining, correlating with the elevated gliosis in these patients. Double-labeling analyses revealed that this elevated CB₂ receptor immunostaining was located in GFAP-labelled astroglial cells. However, we also found CB₂ receptor labeling in cortical neurons confirmed with double immunofluorescence with the neuronal marker MAP-2. This was also found in the spinal cord, using double-labeling with the spinal motor neuron marker choline-acetyl transferase. This happened in both patients and controls, despite these neurons experienced an important degeneration in patients reflected in reduced Nissl staining, TDP-43 immunostaining and CB₁ receptor levels measured by Western blotting. Conclusion: We have confirmed that CB₂ receptors are elevated in the motor cortex of MND patients associated with the reactive gliosis. This phenomenon is previous to neuronal losses. We also found CB₂ receptors in cortical and spinal motor neurons. These observations support that targeting this receptor may serve for developing neuroprotective therapies in MNDs.

Keywords:

• Motor neuron disease
• amyotrophic lateral sclerosis
• post-mortem tissues
• CB1 and CB2 receptors
• FAAH and MAGL enzymes

Declaration of interest

The authors declare that they have no conflicts of interest.

This work has been supported by grants from CIBERNED (CB06/05/0089) and MINECO (SAF2012/39173 and SAF2015-68580-C2-1-R). These agencies had no further role in study design, the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication. Francisco Espejo-Porras is a predoctoral fellow supported by the MINECO (FPI Programme). Authors are indebted to Yolanda García-Movellán for administrative assistance. This work was also supported by Instituto de Salud Carlos III; Secretaría de Estado de Investigación, Desarrollo e Innovación.

Supplementary materials available online