The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

Forebrain GABAergic neurons, the main inhibitory type of neuron in the cortex and hippocampus, represent a highly heterogeneous cell population that has been implicated in the predisposition to epilepsy and the onset of seizure. Earlier attempts to restore inhibition and reduce seizure in animal models of epilepsy have been carried out using embryonic basal forebrain tissue as source of immature GABAergic progenitors in cell-based therapies, with promising results. For therapeutic strategies this approach appears unrealistic, while the use of pluripotent stem cells to obtain immature GABAergic neurons opens new and promising avenues. Research on neural stem cells and pluripotent stem cells has greatly advanced and protocols have been established to efficiently direct progenitor cells to differentiate towards the GABAergic lineage. However, being highly heterogeneous, these neurons are difficult to be fully represented in vitro. Better knowledge on the expressed gene profiles, at single cell level, and the differentiation trajectory of these neurons will consent a more precise monitoring of the differentiation steps. Here we review the current literature about how to obtain and characterize genuine inhibitory neurons, how these can be grafted in animal models (and one day possibly in human) and which diseases could potentially be targeted and the efficiency of therapeutic outcome. The main obstacles that need to be overcome are: a) choice of an appropriate animal model, b) availability of human cells prone to GABA differentiation, c) the full representation of all IN subtypes, their proportions and their physiological activities, d) how to monitor them on the long-term after transplant.

Acrofrontofacionasal Dysostosis type 1 (AFFND1) is a rare human syndrome (estimated prevalence lower than 1:1,000,000), characterized by bone abnormalities in addition to other multiple congenital anomalies and intellectual disability. Only four AFFND1 families, three of Brazilian and one of Indian origin, have been described so far and an autosomal recessive pattern of inheritance has been suggested. The patients are severely affected: the main clinical features are intellectual disability, short stature, facial and skeletal abnormalities characterized by cleft lip/palate, campto-brachy-poly-syndactyly and marked anomalies of foot structures. Exome sequencing in the Indian family, comprising two affected siblings and their parents identified a putative candidate variant in an intronic region of the Neuroblastoma Amplified Sequence (NBAS) gene. This variant was confirmed by Sanger sequencing in the patients and their parents. Since it was located close to an acceptor splice site and predicted to impact on the splicing process, its effect was verified on the cDNA of the patients, and indeed this genomic change was found to cause skipping of exon 48 and premature termination of transcription. Evaluation of expression of the NBAS gene in the mouse osteoblast lineage by semiquantitative RT-PCR showed that NBAS expression was almost completely absent in undifferentiated murine Mesenchymal Stem Cells (mMSCs), but greatly increased in mature osteoblasts derived from them. In addition, immunohistochemical analysis of NBAS during murine fetal life starting at embryonic day (E) 11.5 (that is, when skeletal elements begin to form) showed a pattern of expression in agreement with the multiple abnormalities presented by AFFND1 patients in keeping with its possible role in developmental processes. Further investigations are ongoing in order to unravel the role of the NBAS gene in the pathogenesis of AFFND1 and in general in developmental pathophysiology.