Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin

Neocortical neurons have highly branched dendritic trees that are essential for their function. Indeed, defects in dendritic arborization are associated with human neurodevelopmental disorders. The molecular mechanisms regulating dendritic arbor complexity, however, are still poorly understood. Here, we uncover the molecular basis for the regulation of dendritic branching during cortical development. We show that during development, dendritic branching requires post-mitotic suppression of the RhoGTPase Cdc42. By generating genetically modified mice, we demonstrate that this is catalyzed in vivo by the novel Cdc42-GAP NOMA-GAP. Loss of NOMA-GAP leads to decreased neocortical volume, associated specifically with profound oversimplification of cortical dendritic arborization and hyperactivation of Cdc42. Remarkably, dendritic complexity and cortical thickness can be partially restored by genetic reduction of post-mitotic Cdc42 levels. Furthermore, we identify the actin regulator cofilin as a key regulator of dendritic complexity in vivo. Cofilin activation during late cortical development depends on NOMA-GAP expression and subsequent inhibition of Cdc42. Strikingly, in utero expression of active cofilin is sufficient to restore postnatal dendritic complexity in NOMA-GAP-deficient animals. Our findings define a novel cell-intrinsic mechanism to regulate dendritic branching and thus neuronal complexity in the cerebral cortex

Interleukin-10 restores glutamate receptor-mediated Ca(2+)-signaling in brain circuits under loss of Sip1 transcription factor.

peer reviewedOBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca(2+)-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. RESULTS: It was found that cortical neurons isolated from homozygous (Sip1(fl/fl)) mice with the Sip1 mutation demonstrate suppressed Ca(2+) signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca(2+) oscillations occurring in all neurons of the network in response to Mg(2+)-free medium and bicuculline. But cortical Sip1(fl/fl) neurons only single Ca(2+) pulses or attenuated Ca(2+) oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1(fl/fl) neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1(fl/fl) neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca(2+) signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca(2+) oscillations to a Mg(2+)-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins

Mutation in the Sip1 transcription factor leads to a disturbance of the preconditioning of AMPA receptors by episodes of hypoxia in neurons of the cerebral cortex due to changes in their activity and subunit composition. The protective effects of interleukin-10.

peer reviewedThe Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1(fl/fl) mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca(2+)-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10

Ire1α-Regulated Rate of mRNA Translation is Required for Acquisition of Identity and Polarity in Upper Layer Cortical Neurons

Evolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1α, Ire1α, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1α in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1α engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1α in a mechanism of translational control dependent on 5’UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers

Intracellular Neuroprotective Mechanisms in Neuron-Glial Networks Mediated by Glial Cell Line-Derived Neurotrophic Factor.

peer reviewedGlial cell line-derived neurotrophic factor (GDNF) has a pronounced neuroprotective effect in various nervous system pathologies, including ischaemic brain damage and neurodegenerative diseases. In this work, we studied the effect of GDNF on the ultrastructure and functional activity of neuron-glial networks during acute hypoxic exposure, a key damaging factor in numerous brain pathologies. We analysed the molecular mechanisms most likely involved in the positive effects of GDNF. Hypoxia modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). GDNF (1 ng/ml) was added to the culture medium 20 min before oxygen deprivation. Acute hypoxia-induced irreversible changes in the ultrastructure of neurons and astrocytes led to the loss of functional Сa(2+) activity and neural network disruption. Destructive changes in the mitochondrial apparatus and its functional activity characterized by an increase in the basal oxygen consumption rate and respiratory chain complex II activity during decreased stimulated respiration intensity were observed 24 hours after hypoxic injury. At a concentration of 1 ng/ml, GDNF maintained the functional metabolic network activity in primary hippocampal cultures and preserved the structure of the synaptic apparatus and number of mature chemical synapses, confirming its neuroprotective effect. GDNF maintained the normal structure of mitochondria in neuronal outgrowth but not in the soma. Analysis of the possible GDNF mechanism revealed that RET kinase, a component of the receptor complex, and the PI3K/Akt pathway are crucial for the neuroprotective effect of GDNF. The current study also revealed the role of GDNF in the regulation of HIF-1α transcription factor expression under hypoxic conditions

Olig3 regulates early cerebellar development

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development

The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomes ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology

Timed global reorganization of protein synthesis during neocortex neurogenesis at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Development of the brain’s neocortex requires precisely timed and spatially targeted gene expression, but the relationship between mRNA vs. protein synthesis throughout the genome is unknown. We perform a comprehensive analysis of the reactants, synthesis, and products of mRNA translation spanning mouse neocortex neurogenesis. Ribosome number in the cortical plate decreases sharply at mid-neurogenesis during a transition in neuronal subtype specification, shifting the fundamental kinetics of protein synthesis, with mRNA and protein levels frequently divergent. Satb2, which drives an essential neuronal subtype-specific program, is a highly dynamically translated mRNA with surprisingly broad transcription across diverse neuronal lineages. Satb2 protein achieves its neuronal subtype expression through timed regulation by the RNA-binding protein Pumilio2. Thus, the refinement of transcriptional programs by protein synthesis is a widespread feature of neuronal specification. Developmental neocortex translatome data are provided in an open-source resource: https://shiny.mdc-berlin.de/cortexomics/