Shaping the cerebral cortex by cellular crosstalk.

The cerebral cortex is the brain's outermost layer. It is responsible for processing motor and sensory information that support high-level cognitive abilities and shape personality. Its development and functional organization strongly rely on cell communication that is established via an intricate system of diffusible signals and physical contacts during development. Interfering with this cellular crosstalk can cause neurodevelopmental disorders. Here, we review how crosstalk between migrating cells and their environment influences cerebral cortex development, ranging from neurogenesis to synaptogenesis and assembly of cortical circuits.

Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

NF-κB Signaling in Ex-Vivo Mouse Intestinal Organoids.

We describe here a protocol to assess NF-κB activation in ex-vivo organoids generated from mouse intestinal crypts. These structures are maintained in culture as crypt-villus forming organoids. These ex-vivo organoids maintain both self-renewal and multilineage differentiation overtime. We also describe the generation of ex-vivo organoids from Apc-mutated mouse intestinal crypts. Both wild-type and Apc-mutated organoids respond very well to NF-κB-activating signals such as TNFα but not to LPS. The kinetic of NF-κB activation in response to these signals in ex-vivo intestinal organoids is very similar to what we see in 2D cell lines. This protocol provides investigators a powerful tool to assess NF-κB activation in both healthy and transformed intestinal epitheliums maintained in culture as 3D structures.

Transcranial Magnetic Stimulation-Induced Plasticity Mechanisms: TMS-Related Gene Expression and Morphology Changes in a Human Neuron-Like Cell Model.

Transcranial Magnetic Stimulation (TMS) is a form of non-invasive brain stimulation, used to alter cortical excitability both in research and clinical applications. The intermittent and continuous Theta Burst Stimulation (iTBS and cTBS) protocols have been shown to induce opposite after-effects on human cortex excitability. Animal studies have implicated synaptic plasticity mechanisms long-term potentiation (LTP, for iTBS) and depression (LTD, for cTBS). However, the neural basis of TMS effects has not yet been studied in human neuronal cells, in particular at the level of gene expression and synaptogenesis. To investigate responses to TBS in living human neurons, we differentiated human SH-SY5Y cells toward a mature neural phenotype, and stimulated them with iTBS, cTBS, or sham (placebo) TBS. Changes in (a) mRNA expression of a set of target genes (previously associated with synaptic plasticity), and (b) morphological parameters of neurite outgrowth following TBS were quantified. We found no general effects of stimulation condition or time on gene expression, though we did observe a significantly enhanced expression of plasticity genes NTRK2 and MAPK9 24 h after iTBS as compared to sham TBS. This specific effect provides unique support for the widely assumed plasticity mechanisms underlying iTBS effects on human cortex excitability. In addition to this protocol-specific increase in plasticity gene expression 24 h after iTBS stimulation, we establish the feasibility of stimulating living human neuron with TBS, and the importance of moving to more complex human in vitro models to understand the underlying plasticity mechanisms of TBS stimulation.

Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis.

Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.

Elongator controls cortical interneuron migration by regulating actomyosin dynamics.

The migration of cortical interneurons is a fundamental process for the establishment of cortical connectivity and its impairment underlies several neurological disorders. During development, these neurons are born in the ganglionic eminences and they migrate tangentially to populate the cortical layers. This process relies on various morphological changes that are driven by dynamic cytoskeleton remodelings. By coupling time lapse imaging with molecular analyses, we show that the Elongator complex controls cortical interneuron migration in mouse embryos by regulating nucleokinesis and branching dynamics. At the molecular level, Elongator fine-tunes actomyosin forces by regulating the distribution and turnover of actin microfilaments during cell migration. Thus, we demonstrate that Elongator cell-autonomously promotes cortical interneuron migration by controlling actin cytoskeletal dynamics.

Real-time Recordings of Migrating Cortical Neurons from GFP and Cre Recombinase Expressing Mice.

The cerebral cortex is one of the most intricate regions of the brain that requires elaborate cell migration patterns for its development. Experimental observations show that projection neurons migrate radially within the cortical wall, whereas interneurons migrate along multiple tangential paths to reach the developing cortex. Tight regulation of the cell migration processes ensures proper positioning and functional integration of neurons to specific cerebral cortical circuits. Disruption of neuronal migration often leads to cortical dysfunction and/or malformation associated with neurological disorders. Unveiling the molecular control of neuron migration is thus fundamental to understanding the physiological or pathological development of the cerebral cortex. In this unit, protocols allowing detailed analysis of patterns of migration of both interneurons and projection neurons under different experimental conditions (i.e., loss or gain of function) are presented.