Molecular determinants of endocannabinoid-mediated brain development

The enchanting process of brain development entails a fragile and finite time window, throughout which biochemical imbalances and external insults can potentially affect developing neuronal circuits. Within the numerous exquisitely orchestrated events taking place in the foetal brain, axonal pathfinding is of paramount importance. If aberrant, axonal outgrowth can lead to errors in synaptogenesis and impaired connectivity, with potential long-lasting behavioural phenotypes. Despite the prominent role of endocannabinoids (eCBs) in axonal guidance, the molecular determinants of eCB-mediated axonal regulation remain largely unexplored.

Aiming to interrogate the eCB machinery in less explored subcortical territories, study I investigates the role of the eCB system and its underlying components in foetal cholinergic projection neurons. Prenatal manipulation of cannabinoid receptor 1 (CB1R) permanently reshaped septo-hippocampal cholinergic projections. Nerve growth factor (NGF) was identified as an upstream molecule capable of regulating 2-arachydonoylglycerol (2-AG) via monoacylglycerol lipase (MAGL) degradation. The compartmentalization of each eCB machinery component along extending neurites is therefore crucial. However, axonal pathfinding takes place within the extracellular matrix, migrating glia and developing oligodendrocytes, generating a convoluted scenario where each growth cone interacts with multiple guidance proteins.

Cannabinoid receptor 2 (CB2R)-expressing oligodendrocytes are introduced in study II as an additional player able to drive aberrant callosal axons spread upon supra-physiological 2-AG levels. Excess 2-AG engaged CB2R-mediated premature proliferation of oligodendrocyte end-feet. The interaction between the oligodendrocyte-derived chemorepellant molecule Slit2 and its receptor roundabout 1 (Robo1) on corticofugal axonal ends induced errant CB1R-expressing corticofugal axon pathfinding. In addition to being an endogenous receptor of eCBs, CB1R is also a target for the principal psychoactive compound of cannabis, ∆9-tetrahydrocannabinol (THC).

In study III we undertook an unbiased proteomics screening of the embryonic cortical plate following maternal THC exposure. Superior Cervical Ganglion 10 (SCG10), a microtubule-binding protein present in extending growth cones, was identified as a direct molecular target of THC. Thus, microtubule dynamics represent a novel target of prenatal THC exposure, whose impairment promotes axonal defects and long-lasting synaptic connectivity impairment. Cholecystokinin (Cck)-containing interneurons, due to their high expression of CB1Rs and early appearance in the developing cortex, are likely targets of in utero eCB imbalances. Taking advantage of the recent CckBAC/DsRed mouse line in study IV we achieved a systematic anatomical and physiological characterization of Cck-CB1R-expressing cells from embryonic day (E)10.5 to adulthood.

In sum, multiple upstream and downstream molecular components of the eCBs system were assessed over the course of this thesis investigation, within both physiological brain development and upon maternal cannabis exposure.

List of scientific papers

I. Nerve growth factor scales endocannabinoid signalling by regulating monoacylglycerol lipase turnover in developing cholinergic neurons. Keimpema E, Tortoriello G, Alpár A, Capsoni S, Arisi I, Calvigioni D, Hu SS, Cattaneo A, Doherty P, Mackie K, Harkany T. PNAS. 2013.
https://doi.org/10.1073/pnas.1212563110

II. Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling. Alpár A, Tortoriello G, Calvigioni D, Niphakis MJ, Milenkovic I, Bakker J, Cameron GA, Hanics J, Morris CV, Fuzik J, Kovacs GG, Cravatt BF, Parnavelas JG, Andrews WD, Hurd YL, Keimpema E, Harkany T. Nature Communication. 2014.
https://doi.org/10.1038/ncomms5421

III. Miswiring the brain: Δ9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway. Tortoriello G, Morris CV, Alpar A, Fuzik J, Shirran SL, Calvigioni D, Keimpema E, Botting CH, Reinecke K, Herdegen T, Courtney M, Hurd YL, Harkany T. EMBO J. 2014.
https://doi.org/10.1002/embj.201386035

IV. Functional differentiation of cholecystokinin-containing interneurons destined for the cerebral cortex. Calvigioni D, Máté Z, Fuzik J, Girach F, Zhang MD, Varro A, Beiersdorf J, Schwindling C, Yanagawa Y, Dockray GJ, McBain CJ, Hökfelt T, Szabó G, Keimpema E, Harkany T. Cerebral Cortex. 2016.
https://doi.org/10.1093/cercor/bhw094