In The Spotlight
We read an interesting paper! Here is what it says...about ATP-mediated signalling in the brain
Written by Isabelle
Brain development is a complex and precise process led by a carefully orchestrated genetic program and precise environmental cues. During brain development, neural cells (neurons, astrocytes, and oligodendrocytes) are generated and need to be guided through various developmental stages, which may include proliferation, cell fate determination, migration, maturation, synapse formation, network implementation, and apoptosis. Adenosine Triphosphate (ATP) and other nucleotides are signalling cues essential for directing cells in such crucial developmental steps by mediating intercellular communication. Felipe Ortega and his coworkers at the Universidad Complutense de Madrid collaborated with researchers in other Spanish institutions to investigate whether the ATP/P2X7 receptor purinergic signalling pathway regulates the development of the central nervous system (CNS). They made a quite surprising discovery and they published their results in January 2021 (Ortega et al., Brain Struct Func 2021).
The lack of specific and reliable technical and pharmacological approaches have classically been a major hurdle in the study of purinergic receptors.
The P2X7 receptor (P2X7R) is a purinergic ionotropic receptor with a low affinity to ATP and is permeable to cations (Na+, K+, and Ca2+).There are 10 splice variants of the P2x7r gene that allow for different interactions with intracellular proteins. The receptor’s intracellular domain interacts with various channel dilation mechanisms which could regulate crucial aspects of neuronal cell biology before and after CNS maturation. For instance, P2X7R has been associated with the induction of apoptosis (North RA, Physiol Rev 2002) and thus, it may play a role in the developmentally programmed cell death. In addition, P2X7R could play a role in axonal growth and guidance, cell proliferation, neurotransmitter release, and neuro-inflammation (Miras-Portugal, J Neurosci 2017).
To precisely and reliably investigate the expression pattern of P2X7R, the authors used embryos from a transgenic mouse line, in which the fluorescent protein enhanced-GFP (eGFP) is expressed under the control of the P2rx7 promoter (Gong et al, Nature 2003), to detect and analyse the distribution of the receptor. This mouse line is a very powerful tool in overcoming the lack of reliable technical and pharmacological approaches in detecting this receptor. By analysing the expression pattern of P2X7R, they found two unexpected results:
A clear-cut localization to non-choroidal parts of the entire brain roof plate. A strong fluorescence was detected in the entire neural roof plate and related commissures. The roof plate is an embryonic structure essential for the specification of dorsal interneurons. The fluorescent signal was particularly intense in the circumventricular subfornical and the pretectal subcommissural organs of the roof plate. They also observed numerous fibrillar radial arrangements of labelled glial processes in fluorescent regions, forming a sort of palisade.
The presence of P2X7R in circumventricular organs, which are sites of specialised ependymal cells. Circumventricular organs are known to be functionally diverse and exert sensory or secretory functions, thus providing an interface between the bloodstream and the brain, as well as between brain tissue and cerebrospinal fluid (Kiecker, J Anat 2018). Ortega and colleagues observed expression of P2X7R in several of these circumventricular organs . In the roof plate, the receptor was localised in the subfornical and the pretectal subcommissural organs, whereas in the lateral brain wall they observed it in the zona limitans organ. In the basal hypothalamus P2X7R was expressed in the periventricular hypothalamic organ. These sites have been already identified as embryonic secondary organisers and they are known to release morphogens like SHH, WNT3a and WNT8b, whose gradients influence the fate of cells. Surprisingly, P2X7R was also found in a region that was not previously described. The authors detected a small ependymal specialisation that strongly expressed P2rx7-eGFP signal within the tuberal basal hypothalamic region, and they named this novel circumventricular organ: the postarcuate organ.
Furthermore, the authors report a more heterogeneous expression of P2X7R in various neuronal populations and tracts in other brain areas (i.e. telencephalon, hypothalamus, diencephalon, midbrain, hindbrain, and spinal cord).
In conclusion, this study reports for the first time, the full expression pattern of P2X7R in the embryonic mouse brain. The septum, the neural roof plate (except choroidal areas), and the circumventricular organs strongly expresses this purinergic receptor. Remarkably, this work identifies a novel circumventricular organ, named as the postarcuate organ by the authors, of which the functions remain to be elucidated. This comprehensive analysis of the expression of P2X7R contributes further to the understanding of the role of ATP-mediated purinergic signalling in brain development.
We still do not know its function, but these circumventricular areas often act embryonically as sensory nodes that detect and measure different biological signals.
We sat down with Felipe Ortega for an interview so let’s hear what the main author of this work has to tell us. Before becoming an Associate Professor at the Universidad Complutense de Madrid, where he also obtained his PhD, Felipe Ortega spent several years in Germany as a postdoctoral researcher. First in Munich and then in Mainz, he worked on adult neurogenesis and developed a tool for live imaging of adult neural stem cells isolated from the subependymal zone, which allowed him to study how these cells divide to give rise to neurons in the adult rodent brain.
Q: What was your motivation to study the distribution of P2X7 receptors in the mouse embryos?
We have always been interested in the study of the purinergic signalling and their influence on CNS homeostasis. Within the numerous components of the purinergic system, the P2X7 receptor (P2X7R) stands out as one of the most promising targets with therapeutic potential. Concerning our research, we previously observed that the P2X7R is crucial for the survival of cerebellar granule neurons against different pro-apoptotic stimuli such as glutamate excitotoxicity or growth factor withdrawal. Moreover, our group also described this receptor to be involved in axonogenesis and correct synapse formation in hippocampal neurons. Finally, dysregulation in the signalling triggered by this receptor has also been related to several neurodegenerative disorders such as Hungtington or Alzehimer diseases. However, the lack of specific and reliable technical and pharmacological approaches have classically been a major hurdle in the study of purinergic receptors. This hurdle was partially solved thanks to the transgenic P2rx7-EGFP reporter mice, which expresses EGFP immediately downstream of the mouse P2rx7 proximal promoter, allowing a detailed study of its distribution in the CNS. Thus, we decided to apply this mice line to study the distribution of the P2X7R in the CNS development.
Q: You found a region that was not described previously: a circumventricular ependymal specialisation that you named the postarcuate organ (PArcO). How did you decide to name it like this? Is there a nomenclature to follow or were you free to name it as you wished?
We named it as such because it is an area adjacent to the arcuate organ. This work was a collaboration with Prof Luis Puelles, neuroanatomy professor at the Department of Human Anatomy and Psychobiology, University of Murcia (Spain). Prof Puelles is a worldwide expert in neuroanatomy and brain development and we followed his expert advice in how to formally name this new region.
Q: What was your feeling when you discovered it?
We were indeed thrilled to see the relevant presence of the P2X7R throughout the embryonic mouse CNS development. The analysis of the P2rx7-EGFP reporter mice at the different stages of embryonic development has opened new challenging research lines in our group. Overall, discovering and describing the PArcO region was truly outstanding for both us and Prof Puelles, whose contribution was crucial for describing the distribution of the receptor and, undoubtedly, for describing the PArcO.
Q: Could you speculate on the function of the postarcuate organ?
We still do not know its function, but these circumventricular areas often act embryonically as sensory nodes that detect and measure different biological signals, and may react to changes or alterations in the measured variable. Likewise, such specialisations may act as secondary organisers, that is, they may release morphogens that diffuse into neighbouring neural territories, establishing concentration gradients. That could be a good starting point to speculate about PArcO function.
- Ortega et al (2021) Salient brain entities labelled in P2rx7-EGFP reporter mouse embryos include the septum, roof plate glial specializations and circumventricular ependymal organs. Brain Struct Funct. 226(3): 715–741
- North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067
- Miras-Portugal MT, Sebastian-Serrano A, de Diego GL, Diaz-Hernandez M (2017) Neuronal P2X7 Receptor: Involvement in Neuronal Physiology and Pathology. J Neurosci 37:7063–7072
- Gong S et al (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917–925
- Kiecker C (2018) The origins of the circumventricular organs. J. Anat. (2018) 232, pp540--553
Written by Isabelle Arnoux; Edited by Chiara Galante & John (JJ) Fung. Featured Image: NGC/Design.