Identifying astroglial molecular contributors to epileptogenesis

Background

Epilepsy is one of the most common neurological disorders – estimated to affect at least 65 million worldwide. Most epilepsy research has so far focused on how to dampen neuronal discharges. Epileptic tissue is, however, also characterized by pronounced pathological changes affecting astrocytes, the main glial cell of gray matter. Astrocytes across epilepsy entities typically exhibit reactive astrogliosis, which is a universal pathophysiological response of astrocytes involving changes in morphology, gene expression and function¹. The consequences of these pathological processes in astrocytes remain rudimentarily explored.

Aims and research question(s)

Here we AIM to investigate the following research questions: 

1. How does endogenous cannabinoid (endocannabinoid) signaling affect astrocytic Ca²⁺ signaling and the development of reactive astrogliosis in epilepsy?
2. Are the beneficial effects of (exogenous) cannabinoids due to modulation of reactive astrogliosis?

Esguerra et al. have established a potent model of Dravet syndrome (DS)², a disorder most commonly caused by a mutation in SCN1A, encoding a voltage gated sodium channel, that causes severe epilepsy and cognitive impairment. They generated and studied zebrafish with an scn1a loss of function mutation using single cell transcriptomics. Remarkably, we observed a profound early reactive astrogliosis in their epileptic zebrafish. These findings were corroborated recently by another study on a rodent SCN1A Dravet model³. In physiology, it is now widely accepted that astrocytes are key controllers of the composition of the extracellular fluids and may directly interact with neurons by releasing gliotransmitters. A central tenet is that astrocytic intracellular Ca²⁺ signals promote release of such signaling substances. Remarkably, very little is currently known about how reactive gliosis in epilepsy affects astrocytic Ca²⁺ signalling and the pathophysiology of epilepsy at large. GliaLab (Enger) has presented data suggesting that astrocytic Ca²⁺ signaling is pro-epileptic during acute seizures⁴, and that astrocytes following status epilepticus and in chronic epilepsy exhibit increased astrocytic Ca²⁺ signalling⁵, but could be antiepileptic throughout epileptogenesis (unpublished data).

Endocannabinoid signaling is a potent signaling pathway in astrocytes, and a key trigger of astrocytic Ca²⁺ signalling^6,7. Moreover, cannabinoid treatment has, in recent years, proven to be a highly effective treatment for several types of refractory epilepsies⁸. In 2018, the FDA approved cannabidiol (CBD, Epidiolex, GW Pharmaceuticals) for the treatment of Dravet and Lennox Gastaut syndromes – two catastrophic, drug resistant, early onset epilepsy syndromes. Importantly, cannabidiol has already shown effective seizure reduction in a zebrafish Dravet syndrome model⁹. Evidence suggests that endocannabinoids are produced post-synaptically in hyperexcitable states and serve as retrograde signals that dampen neuronal excitation through the activation of presynaptic CB1 receptors. The effects of CBD treatment are likely in part explained by boosting this negative feedback mechanism. Key CBD targets are expressed in glial cells (e.g., CB1, CB2, PPAR-γ and 5-HT1A) and emerging evidence indicates that part of the proposed CBD modes of action are mediated by interference with glial cell function¹⁰. However, no studies have addressed how endocannabinoid signaling or treatment with cannabinoids could affect reactive astrogliosis and Ca²⁺ signals in reactive astrocytes in intact animals in the development of epilepsy.

Methods

In this study, we will analyze the effect of CBD and structural analogs on astrocyte function in two refractory epilepsy models in vivo – namely the intracortical kainate model in mice and the Dravet syndrome (DS; SCN1a mutant) model in zebrafish: 

• Imaging astrocytes in CBD treated DS larvae: We will use confocal fluorescence microscopy to visualize how CBD modulates astrocyte function in wildtype and DS mutant larvae. We will image neuronal and astrocyte interactions during the epileptogenic period, using transgenic fluorescent reporter lines (astrocytes, GABAergic and Glutamatergic neurons) intercrossed with the fish DS mutant. Emphasis will be placed on determining the exact treatment window and dose regimen required for CBD to modulate astrocyte activity and achieve a positive therapeutic outcome in the DS model (Tiraboschi et al. 2020). We will then visualize Ca²⁺ signaling in astrocytes in DS larvae (+/- CBD). Chemical modifier analyses will be carried out to identify astrocytic target pathways.
• Perturbing astrocytic signaling pathways modulated by CBD: In astrocytes, endocannabinoids likely trigger astrocytic Ca²⁺ signals through activation of IP₃ receptor 2 ^6,7. We aim to investigate the effects of removing IP₃ receptor 2 on the development of astrogliosis and epilepsy phenotype in mice subjected to deep intracortical kainate injection¹¹ by continuous wireless EEG telemetry. If CBD administration affects gliosis in the DS model, we will subject epileptic mice to CBD treatment and monitor their development of an epileptic phenotype. Pending the findings in these investigations, the long-term goal is to also perform in vivo astrocytic and neuronal Ca²⁺ imaging by two-photon microscopy of hippocampal gliosis with or without CBD treatment.
• NOTE: Other drug candidates (both identified in the Esguerra lab and by other groups) shown to inhibit seizures in rodent and fish refractory epilepsy models (with several entering clinical trials) may also be considered for testing in the above-mentioned assays, for comparison with CBD activity.

Students tasks

The student will be supervised by both Rune Enger (GliaLab and the Letten Centre, Institute of Basic Medical Sciences) and Camila Esguerra (Chemical Neuroscience Group, NCMM) and will receive training in both research laboratories. Experiments on rodents will be carried out in the Enger lab, while zebrafish experiments will be performed in the Esguerra lab. The student will benefit from gaining expertise in pre-clinical biomedical research using two model organisms, interactions with the two groups in terms of training from more experienced PhD students and postdoctoral fellows, experimental approach and communication of research results. The student will be trained and developed as an independent researcher and is expected to present and discuss their results at group meetings, as well as learn to provide feedback to other team members. The goal is to present his/her research findings at an international conference towards the latter half of the training. This PhD project is part of a larger research program carried out by both groups, with the common aim of elucidating the molecular mechanisms underlying epileptogenesis.

Contact information

Rune Enger

Camila Vicencio Esguerra

References

1.Escartin, C. et al. Reactive astrocyte nomenclature, definitions, and future directions. Nat. Neurosci. 24, 312–325 (2021).

2. Tiraboschi, E. et al. New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome. Epilepsia 61, 549–560 (2020).

3. Uchino, K. et al. Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/− Mouse Model of Dravet Syndrome. bioRxiv doi:10.1101/2021.05.18.444602.

4. Heuser, K. et al. Ca2+ Signals in Astrocytes Facilitate Spread of Epileptiform Activity. Cereb. Cortex 28, 4036–4048 (2018).

5. Szokol, K. et al. Augmentation of Ca2+ signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy. Front. Cell. Neurosci. 9, 49 (2015).

6. Navarrete, M. et al. Astrocytes in endocannabinoid signalling. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130599 (2014).

7. Navarrete, M. & Araque, A. Endocannabinoids mediate neuron-astrocyte communication. Neuron 57, 883–893 (2008).

8. Morano, A. et al. Cannabinoids in the treatment of epilepsy: Current status and future prospects. Neuropsychiatr. Dis. Treat. 16, 381–396 (2020).

9. Thornton, C. et al., Cannabis constituents reduce seizure behavior in chemically-induced and scn1a-mutant zebrafish. Epilepsy Behav. 110, 107152 (2020).

10. Scarante, F.F. et al., Glial cells and their contribution to the mechanisms of action of cannabidiol in neuropsychiatric disorders. Front. Pharmacol. 11, 618065 (2020).

11. Bedner, P. et al. Astrocyte uncoupling as a cause of human temporal lobe epilepsy. Brain 138, 1208–1222 (2015).