A Cannabidiol-Regulated MicroRNA Controls Brain Excitability and is a Target for Seizure Control

Epilepsy is a serious neurological disease which is primarily characterised by spontaneous recurrent seizures, affecting approximately 65 million people worldwide. So far there is still no pharmacological cure for the disease and the current anti-seizure drugs (ASDs) only treat the symptoms and do not modify the underlying pathophysiology or prevent the progression of the disease. There remains an urgent need for new therapies for drug-resistant epilepsy, as ~30% of affected people do not respond adequately to current pharmacological treatments. In recent years, cannabidiol (CBD) emerged as a new therapy option for several epileptic encephalopathies, including Dravet syndrome and Lennox-Gastaut syndrome. However, the extent to which CBD is anti-seizure and the mechanism(s) by which CBD mediates its anticonvulsant effects are not elucidated. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and have shown to be involved in the pathophysiology of epilepsy. To date, over 20 miRNAs have been functionally investigated in different animal models of epilepsy and seizures and proven to modulate seizure activity or associated pathologies.

The aim of this thesis was to explore whether CBD regulates specific miRNAs and then to use the findings to probe novel actions of miRNAs in the setting of epilepsy. The studies combined in vivo seizure models, small RNA sequencing, in silico microRNA target analysis, ex vivo brain slice electrophysiology and molecular biology. 

The first results chapter characterised the anti-seizure properties of CBD in a set of preclinical models of acquired and genetic epilepsies. As expected, dosing mice with CBD protected mice in the pentylenetetrazol (PTZ) model against seizures, both severity and improved survival. CBD also reduced hyperthermia-induced seizure severity in a mouse model of Dravet syndrome (Scn1atm1Kea). However, CBD treatment did not alter status epilepticus (SE) or the occurrence or severity of spontaneous recurrent seizures in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy. In addition to the observed anti-seizure effects, studies here also determined that CBD has a dose-related hypothermic effect in mice.

The second results chapter investigated whether cannabinoids, in particular CBD, alter the expression of miRNAs in the mouse hippocampus. Using small RNA sequencing, CBD produced significant changes to the hippocampal expression of miRNAs. This effect was more extensive than for the two closely-related phytocannabinoids cannabidivarin (CBDV) and cannabigerol (CBG). MiRNA-335 was identified as an abundant miRNA downregulated after CBD treatment. MiRNA target interaction analysis identified several epilepsy-related miR-335 targets, including voltage-gated sodium channels (VGSCs).

The third results chapter explored how bi-directional modulation of miR-335 affects excitability and the expression of VGSCs. Inhibition of miR-335 using an antimiR (Ant-335) de-repressed various VGSC subtypes in the mouse brain in adult but not young (P21) C57BL/6 mice. Ex vivo brain slice electrophysiology detected changes in the amplitude, and rising phase of action potentials of hippocampal pyramidal cells of Ant-335 treated mice. These changes in the action potential properties were consistent with an effect to increased firing frequency of individual neurons. Furthermore, an AAV9 was designed and deployed to upregulate miR-335 to explore the effects of in vivo overexpression of miR-335 in the brain.

In the final results chapter, functional studies investigated how changes to miR-335 levels affect seizures in a set of different mouse models. Inhibition of miR-335 slightly increased the susceptibility to seizures in the intra-amygdala kainic acid model and PTZ model whereas overexpression of miR-335 reduced the severity of PTZ-induced seizures and improved the survival rate. However, modulation of miR-335 produced variable effects on the expression of VGSCs in the different models. 

In conclusion, these studies provide evidence that CBD may exert some of its effects via modulation of miRNAs. Moreover, we identify miR-335 as an important regulator of brain excitability through network effects on components that set neuronal excitability thresholds. Findings here point also to complex, context-specific bi-directional effects of this miRNA on targets and brain excitability which are age-dependent. Targeting miR-335 may represent a new therapeutic strategy for the epilepsies, including sodium channelopathies.