Publicação
The role of P2 receptors in the migration of medial ganglionic eminence-derived interneurons
| Resumo: | The cerebral cortex circuitry relies essentially on a balanced network between excitatory pyramidal neurons and inhibitory GABAergic interneurons. One of the fundamental processes for the establishment of such correctly wired circuitry is neuronal migration. Indeed, impairment of neuronal migration is among the most common causes of cortical malformation disorders and, particularly, inhibitory interneuron defects have been often associated with several neurological and psychiatric disorders. Thus, it is paramount to unravel the precise mechanisms underlying interneuron migration. In this regard, it was recently shown that purinergic signalling contributes to neuronal migration as it was reported that the pharmacological blockade of adenosine A2A receptors delays migration and insertion of interneurons into the hippocampal circuitry. Moreover, it has also been reported that P2Y1R is crucial for the migration of intermediate neuronal progenitors and proper formation of the cortical SVZ. Besides, it has been recently shown that P2 receptors can bidirectionally modulate neurite outgrowth, a crucial event for neuronal migration. This has prompted the study of the role of purinergic receptors in interneuron migration. Hence, the main aim of this work was to identify and characterize the role of P2 receptors in the migration of interneurons derived from the MGE, the main source of cortical GABAergic interneurons. It was found that P2Y1R is expressed in the brain at mid-late stages of embryogenesis (E12.5-onwards), coincident with the onset of interneuron migration, and particularly in MGE-derived interneurons (E13.5). Its presence was functionally appraised by the observation of intracellular Ca2+ transients induced by a selective agonist of P2Y1R, MRS2365 (100 nM). By using an ex vivo model consisting in 3D cultures of MGE explants from E13.5 mice brains, it was observed that the blockade of P2Y1R with the selective antagonists MRS2179 (10 µM) or MRS2500 (10 µM) significantly decreased the migration of interneurons from the MGE explants. This effect was mimicked by apyrase (20 U/mL), an enzyme that catabolizes ATP and ADP into AMP, being the migration restored upon the pharmacological activation of P2Y1R with MRS2365 (100 nM). This effect is mediated by PKC as it was arrested by the presence of a PKC inhibitor (BIM-1, 500 nM). Therefore, these data show for the first time that P2Y1R is expressed and tonically promotes MGE-derived interneuron migration through PKC activation. In contrast, the P2X-preferring agonist BzATP (1, 10, 100 µM) inhibited MGE-derived interneuron migration in a concentration-dependent manner, an effect prevented by the blockade of P2 receptors (PPADS, 10 µM), by the selective blockade of P2X1R (NF279, 1 µM), but not modified by the selective P2X7R antagonist A438079 (10 µM). These results demonstrate that P2X1R inhibits MGE-derived interneuron migration. Although the inhibitory effect of BzATP on interneuron migration is not mediated by P2X7R, it was also observed that P2X7R is tonically contributing to the migration of MGE-derived interneurons based on the observation that the P2X7R blockade with two different selective antagonists, A438079 (10 µM) and A740003 (10 µM), inhibited interneuron migration. At the cellular level, it was gathered evidence indicating that P2Y1R may be promoting interneuron migration by contributing to leading process branching, a critical step in the saltatory movement cycle. Instead, P2X1R-induced inhibition of MGE-derived interneuron migration should be caused by disruption of the formation of new leading process branches affecting cell orientation, utterly suggesting a relationship between P2 receptors and cytoskeleton dynamics. This is supported by the expression and localization of P2Y1R and P2X1R in the leading processes of MGE cells, including at the most distal end. Conversely, P2X7R expression in MGE interneurons is restricted to the soma and proximal part of the leading process, which is in accordance with its lack of involvement on the BzATP-induced inhibition of migration. Furthermore, it was also provided evidence suggesting that the regulation of leading process dynamics by P2Y1R may be attained by the modulation of CRMP2 phosphorylation, a microtubule-associated protein that plays a crucial role in axonal guidance and outgrowth. The selective blockade of P2Y1R (MRS2179, 10 µM) increased the inhibitory phosphorylation of CRMP2T514 most likely reflecting the observed decrease of the inhibitory phosphorylation of GSK3βS9. These modifications were accompanied by a reduction of the number and size of growth cones in MGE cells, which may be due to induced growth cone collapse. Thus, these observations indicate that P2Y1R may regulate leading process dynamics and the migration of MGE-derived interneurons through GSK3β-CRMP2 pathway. This work further shows that immunoreactivity for P2X1R in the E13.5 embryonic telencephalon is mostly found in βIII-tubulin-positive migratory neurons in contrast to the predominant expression of P2Y1R in proliferative zones (βIII-tubulin-negative) including in the MGE. Besides, it was shown that P2X1R impairs cell orientation of MGE-derived interneurons, in opposition of P2Y1R that does not influence their guidance. Together, this evidence supports a scenario in which P2Y1R promotes interneuron migration as a motogenic factor in the MGE, while the P2X1R should be contributing for their guidance by transducing ATP as a repulsive cue. Indeed, it was observed that P2Y1R and P2XRs control neuronal migration of MGE-derived interneurons in a differential time-dependent manner, wherein the contribution of P2Y1R activity seems particularly relevant in the initial phase of migration, while the ability of P2XR activation to inhibit migration is kept at later periods of migration. This supports the postulated hypothesis of P2Y1R acting as a motogenic receptor to promote the initial migration of cells away from the proliferative zones, and P2X1R as a guidance receptor in the control of MGE-derived interneuron migration. Overall, this study provides compelling evidence that rise P2 receptors as novel players in the control of interneuron migration during development and re-enforces for the pivotal role of purinergic signalling in neuronal migration and in corticogenesis. |
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| Autores principais: | Ferreira, Sofia Alexandra Ramos |
| Assunto: | ATP corticogenesis CRMP2 interneuron migration leading process branching medial ganglionic eminence orientation purinergic signalling P2Y1 receptor P2X1 receptor P2X7 receptor |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | The cerebral cortex circuitry relies essentially on a balanced network between excitatory pyramidal neurons and inhibitory GABAergic interneurons. One of the fundamental processes for the establishment of such correctly wired circuitry is neuronal migration. Indeed, impairment of neuronal migration is among the most common causes of cortical malformation disorders and, particularly, inhibitory interneuron defects have been often associated with several neurological and psychiatric disorders. Thus, it is paramount to unravel the precise mechanisms underlying interneuron migration. In this regard, it was recently shown that purinergic signalling contributes to neuronal migration as it was reported that the pharmacological blockade of adenosine A2A receptors delays migration and insertion of interneurons into the hippocampal circuitry. Moreover, it has also been reported that P2Y1R is crucial for the migration of intermediate neuronal progenitors and proper formation of the cortical SVZ. Besides, it has been recently shown that P2 receptors can bidirectionally modulate neurite outgrowth, a crucial event for neuronal migration. This has prompted the study of the role of purinergic receptors in interneuron migration. Hence, the main aim of this work was to identify and characterize the role of P2 receptors in the migration of interneurons derived from the MGE, the main source of cortical GABAergic interneurons. It was found that P2Y1R is expressed in the brain at mid-late stages of embryogenesis (E12.5-onwards), coincident with the onset of interneuron migration, and particularly in MGE-derived interneurons (E13.5). Its presence was functionally appraised by the observation of intracellular Ca2+ transients induced by a selective agonist of P2Y1R, MRS2365 (100 nM). By using an ex vivo model consisting in 3D cultures of MGE explants from E13.5 mice brains, it was observed that the blockade of P2Y1R with the selective antagonists MRS2179 (10 µM) or MRS2500 (10 µM) significantly decreased the migration of interneurons from the MGE explants. This effect was mimicked by apyrase (20 U/mL), an enzyme that catabolizes ATP and ADP into AMP, being the migration restored upon the pharmacological activation of P2Y1R with MRS2365 (100 nM). This effect is mediated by PKC as it was arrested by the presence of a PKC inhibitor (BIM-1, 500 nM). Therefore, these data show for the first time that P2Y1R is expressed and tonically promotes MGE-derived interneuron migration through PKC activation. In contrast, the P2X-preferring agonist BzATP (1, 10, 100 µM) inhibited MGE-derived interneuron migration in a concentration-dependent manner, an effect prevented by the blockade of P2 receptors (PPADS, 10 µM), by the selective blockade of P2X1R (NF279, 1 µM), but not modified by the selective P2X7R antagonist A438079 (10 µM). These results demonstrate that P2X1R inhibits MGE-derived interneuron migration. Although the inhibitory effect of BzATP on interneuron migration is not mediated by P2X7R, it was also observed that P2X7R is tonically contributing to the migration of MGE-derived interneurons based on the observation that the P2X7R blockade with two different selective antagonists, A438079 (10 µM) and A740003 (10 µM), inhibited interneuron migration. At the cellular level, it was gathered evidence indicating that P2Y1R may be promoting interneuron migration by contributing to leading process branching, a critical step in the saltatory movement cycle. Instead, P2X1R-induced inhibition of MGE-derived interneuron migration should be caused by disruption of the formation of new leading process branches affecting cell orientation, utterly suggesting a relationship between P2 receptors and cytoskeleton dynamics. This is supported by the expression and localization of P2Y1R and P2X1R in the leading processes of MGE cells, including at the most distal end. Conversely, P2X7R expression in MGE interneurons is restricted to the soma and proximal part of the leading process, which is in accordance with its lack of involvement on the BzATP-induced inhibition of migration. Furthermore, it was also provided evidence suggesting that the regulation of leading process dynamics by P2Y1R may be attained by the modulation of CRMP2 phosphorylation, a microtubule-associated protein that plays a crucial role in axonal guidance and outgrowth. The selective blockade of P2Y1R (MRS2179, 10 µM) increased the inhibitory phosphorylation of CRMP2T514 most likely reflecting the observed decrease of the inhibitory phosphorylation of GSK3βS9. These modifications were accompanied by a reduction of the number and size of growth cones in MGE cells, which may be due to induced growth cone collapse. Thus, these observations indicate that P2Y1R may regulate leading process dynamics and the migration of MGE-derived interneurons through GSK3β-CRMP2 pathway. This work further shows that immunoreactivity for P2X1R in the E13.5 embryonic telencephalon is mostly found in βIII-tubulin-positive migratory neurons in contrast to the predominant expression of P2Y1R in proliferative zones (βIII-tubulin-negative) including in the MGE. Besides, it was shown that P2X1R impairs cell orientation of MGE-derived interneurons, in opposition of P2Y1R that does not influence their guidance. Together, this evidence supports a scenario in which P2Y1R promotes interneuron migration as a motogenic factor in the MGE, while the P2X1R should be contributing for their guidance by transducing ATP as a repulsive cue. Indeed, it was observed that P2Y1R and P2XRs control neuronal migration of MGE-derived interneurons in a differential time-dependent manner, wherein the contribution of P2Y1R activity seems particularly relevant in the initial phase of migration, while the ability of P2XR activation to inhibit migration is kept at later periods of migration. This supports the postulated hypothesis of P2Y1R acting as a motogenic receptor to promote the initial migration of cells away from the proliferative zones, and P2X1R as a guidance receptor in the control of MGE-derived interneuron migration. Overall, this study provides compelling evidence that rise P2 receptors as novel players in the control of interneuron migration during development and re-enforces for the pivotal role of purinergic signalling in neuronal migration and in corticogenesis. |
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