CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder with unclear underlying mechanisms. Here, the authors unravel the contribution of a stress-responsive pathway to RSTS where impaired HSF2 acetylation, due to RSTS-associated CBP/EP300 mutations, alters the expression of neurodevelopmental players, in keeping with hallmarks of cell-cell adhesion defects.Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.</p

Caracterització dels ratolins knockin condicional de PDK1 que expressen la mutació L155E a sistema nerviós central

La via de la PI3K/PKB juga un paper molt important en el sistema nerviós central durant el desenvolupament neuronal. PDK1 és la màster quinasa que s’encarrega de coordinar les diverses senyals de la PI3K, regulant diferencialment l’activació de diferents AGC quinases. A fi d’estudiar el paper de PDK1 al sistema nerviós central, vàrem generar dos models de ratolí que presentaven mutats dos dominis funcionals d’aquesta quinasa: el PH-domain i el PIF-pocket. En aquesta tesi es desenvolupa la caracterització del model de ratolí knock-in condicional que expressa la mutació L155E de PDK1 a sistema nerviós central. Aquesta mutació resulta en la inhabilitació del domini PIF-pocket de PDK1 i per tant en una disrupció del mecanisme d’activació de totes les AGC quinases que no presenten PH-domain, com són RSK, SGK i S6K, però no PKB. La manca d’activació d’aquestes quinases no resulta essencial per les respostes de supervivència i viabilitat cel·lular, però sí que impedeix el correcte desenvolupament neuronal, el que es tradueix en un dèficit de la polarització i diferenciació neuronal. Els defectes en aquests processos neuronals resulten en una reducció de la mida cerebral i en alteracions fisiològiques de regions del cervell com el còrtex o l’hipocamp. Aquestes alteracions impliquen una reducció en la connectivitat neuronal i en la comunicació entre interneurones gabaèrgiques i neurones piramidals, possiblement causada per dèficits en els processos de migració durant el desenvolupament. Tot això condueix a un model de ratolí que presenta alterada la seva conducta davant l’estrès i emula els símptomes negatius i cognitius de trastorns psiquiàtrics com l’esquizofrènia. Aquests resultats mostren la rellevància de PDK1, orquestrant la via de la PI3K, tant durant el desenvolupament neuronal com en la consolidació del correcte funcionament del cervell.The PI3K/PKB signalling pathway plays an indispensable role in the central nervous system during neuronal development. PDK1 is the master kinase which orchestrates the PI3K signals by regulating the activation of AGC kinases. In order to elucidate the role of PDK1 in the central nervous system, two conditional knockin mice models were generated based on the disruption of two functional domains of PDK1 that are essential for substrate activation: the PH-domain and the PIF-pocket. In this thesis, I generated and characterized the brain-specific PDK1 conditional knockin mice which express the L155E mutation within the nervous system. This mutation disrupts the PDK1 PIF-pocket domain impairing the activation of RSK, SGK and S6K, whereas PKB activation was not affected. Deficits in the PIF-pocket dependent kinases activation were not translated into a significant deregulation of the neuronal survival, whilst neuronal polarization and differentiation were severely impaired. The abnormal neuronal development resulted in microcephaly as well as a huge neuronal circuitry reduction in the cortex and hippocampus brain areas. These physiological alterations broke the communication between the gabaergic and the pyramidal neurons, probably due to a deregulation in the neuronal migration process. Mice expressing the PDK1 L155E mutation in the central nervous system were vulnerable in front of stress insults, with behavioural outputs that reproduced a new model for negative and cognitive symptoms related to psychiatric disorders such as schizophrenia. Altogether, these studies show us the relevance of PDK1, acting as the master kinase involved in PI3K signalling pathway regulation, in neuronal development leading to the appropriate consolidation of cerebral functions

Caracterització dels ratolins knockin condicional de PDK1 que expressen la mutació L155E a sistema nerviós central

La via de la PI3K/PKB juga un paper molt important en el sistema nerviós central durant el desenvolupament neuronal. PDK1 és la màster quinasa que s'encarrega de coordinar les diverses senyals de la PI3K, regulant diferencialment l'activació de diferents AGC quinases. A fi d'estudiar el paper de PDK1 al sistema nerviós central, vàrem generar dos models de ratolí que presentaven mutats dos dominis funcionals d'aquesta quinasa: el PH-domain i el PIF-pocket. En aquesta tesi es desenvolupa la caracterització del model de ratolí knock-in condicional que expressa la mutació L155E de PDK1 a sistema nerviós central. Aquesta mutació resulta en la inhabilitació del domini PIF-pocket de PDK1 i per tant en una disrupció del mecanisme d'activació de totes les AGC quinases que no presenten PH-domain, com són RSK, SGK i S6K, però no PKB. La manca d'activació d'aquestes quinases no resulta essencial per les respostes de supervivència i viabilitat cel·lular, però sí que impedeix el correcte desenvolupament neuronal, el que es tradueix en un dèficit de la polarització i diferenciació neuronal. Els defectes en aquests processos neuronals resulten en una reducció de la mida cerebral i en alteracions fisiològiques de regions del cervell com el còrtex o l'hipocamp. Aquestes alteracions impliquen una reducció en la connectivitat neuronal i en la comunicació entre interneurones gabaèrgiques i neurones piramidals, possiblement causada per dèficits en els processos de migració durant el desenvolupament. Tot això condueix a un model de ratolí que presenta alterada la seva conducta davant l'estrès i emula els símptomes negatius i cognitius de trastorns psiquiàtrics com l'esquizofrènia. Aquests resultats mostren la rellevància de PDK1, orquestrant la via de la PI3K, tant durant el desenvolupament neuronal com en la consolidació del correcte funcionament del cervell.The PI3K/PKB signalling pathway plays an indispensable role in the central nervous system during neuronal development. PDK1 is the master kinase which orchestrates the PI3K signals by regulating the activation of AGC kinases. In order to elucidate the role of PDK1 in the central nervous system, two conditional knockin mice models were generated based on the disruption of two functional domains of PDK1 that are essential for substrate activation: the PH-domain and the PIF-pocket. In this thesis, I generated and characterized the brain-specific PDK1 conditional knockin mice which express the L155E mutation within the nervous system. This mutation disrupts the PDK1 PIF-pocket domain impairing the activation of RSK, SGK and S6K, whereas PKB activation was not affected. Deficits in the PIF-pocket dependent kinases activation were not translated into a significant deregulation of the neuronal survival, whilst neuronal polarization and differentiation were severely impaired. The abnormal neuronal development resulted in microcephaly as well as a huge neuronal circuitry reduction in the cortex and hippocampus brain areas. These physiological alterations broke the communication between the gabaergic and the pyramidal neurons, probably due to a deregulation in the neuronal migration process. Mice expressing the PDK1 L155E mutation in the central nervous system were vulnerable in front of stress insults, with behavioural outputs that reproduced a new model for negative and cognitive symptoms related to psychiatric disorders such as schizophrenia. Altogether, these studies show us the relevance of PDK1, acting as the master kinase involved in PI3K signalling pathway regulation, in neuronal development leading to the appropriate consolidation of cerebral functions

Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits

A high percentage of patients with brain metastases frequently develop neurocognitive symptoms; however, understanding how brain metastasis co-opts the function of neuronal circuits beyond a tumor mass effect remains unknown. We report a comprehensive multidimensional modeling of brain functional analyses in the context of brain metastasis. By testing different preclinical models of brain metastasis from various primary sources and oncogenic profiles, we dissociated the heterogeneous impact on local field potential oscillatory activity from cortical and hippocampal areas that we detected from the homogeneous inter-model tumor size or glial response. In contrast, we report a potential underlying molecular program responsible for impairing neuronal crosstalk by scoring the transcriptomic and mutational profiles in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help predict the presence and subtype of metastasi

CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

International audienceAbstract Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology

Stress-induced unfolded protein response contributes to Zika virus-associated microcephaly.

Accumulating evidence support a causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. However, the mechanism of ZIKV-associated microcephaly remains unclear. We combined analyses of ZIKV-infected human fetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show that ZIKV triggers endoplasmic reticulum stress and unfolded protein response in the cerebral cortex of infected postmortem human fetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained endoplasmic reticulum stress leads to apoptosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of unfolded protein response counteracts these pathophysiological mechanisms and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV, as they are not observed upon intraplacental injection of other related flaviviruses in mice