LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) ß-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

Studying human neural function in vivo at the cellular level: Chasing chimeras?

Despite its importance to understanding human brain (dys)function, it has remained challenging to study human neurons in vivo. Recent approaches, using transplantation of human cortical neurons into the rodent brain, offer new prospects for the study of human neural function and disease in vivo, from molecular to circuit levels.

Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta/Notch Regulation.

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution. Gene duplications constitute a major evolutionary force, but their impact on human brain development remains unclear. Using tailored RNA sequencing (RNA-seq), we profiled the spatial and temporal expression of hominid-specific duplicated (HS) genes in the human fetal cortex and identified a repertoire of 35 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among them NOTCH2NL, human-specific paralogs of the NOTCH2 receptor, stood out for their ability to promote cortical progenitor maintenance. NOTCH2NL promote the clonal expansion of human cortical progenitors, ultimately leading to higher neuronal output. At the molecular level, NOTCH2NL function by activating the Notch pathway through inhibition of cis Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes active during human corticogenesis and reveals how human-specific NOTCH paralogs may have contributed to the expansion of the human cortex.

Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

Cellular and Molecular Mechanisms Linking Human Cortical Development and Evolution.

The cerebral cortex is at the core of brain functions that are thought to be particularly developed in the human species. Human cortex specificities stem from divergent features of corticogenesis, leading to increased cortical size and complexity. Underlying cellular mechanisms include prolonged patterns of neuronal generation and maturation, as well as the amplification of specific types of stem/progenitor cells. While the gene regulatory networks of corticogenesis appear to be largely conserved among all mammals including humans, they have evolved in primates, particularly in the human species, through the emergence of rapidly divergent transcriptional regulatory elements, as well as recently duplicated novel genes. These human-specific molecular features together control key cellular milestones of human corticogenesis and are often affected in neurodevelopmental disorders, thus linking human neural development, evolution, and diseases.

Developmental mechanisms underlying the evolution of human cortical circuits.

The brain of modern humans has evolved remarkable computational abilities that enable higher cognitive functions. These capacities are tightly linked to an increase in the size and connectivity of the cerebral cortex, which is thought to have resulted from evolutionary changes in the mechanisms of cortical development. Convergent progress in evolutionary genomics, developmental biology and neuroscience has recently enabled the identification of genomic changes that act as human-specific modifiers of cortical development. These modifiers influence most aspects of corticogenesis, from the timing and complexity of cortical neurogenesis to synaptogenesis and the assembly of cortical circuits. Mutations of human-specific genetic modifiers of corticogenesis have started to be linked to neurodevelopmental disorders, providing evidence for their physiological relevance and suggesting potential relationships between the evolution of the human brain and its sensitivity to specific diseases.

Inhibition of SRGAP2 function by its human-specific paralogs induces neoteny during spine maturation.

Structural genomic variations represent a major driving force of evolution, and a burst of large segmental gene duplications occurred in the human lineage during its separation from nonhuman primates. SRGAP2, a gene recently implicated in neocortical development, has undergone two human-specific duplications. Here, we find that both duplications (SRGAP2B and SRGAP2C) are partial and encode a truncated F-BAR domain. SRGAP2C is expressed in the developing and adult human brain and dimerizes with ancestral SRGAP2 to inhibit its function. In the mouse neocortex, SRGAP2 promotes spine maturation and limits spine density. Expression of SRGAP2C phenocopies SRGAP2 deficiency. It underlies sustained radial migration and leads to the emergence of human-specific features, including neoteny during spine maturation and increased density of longer spines. These results suggest that inhibition of SRGAP2 function by its human-specific paralogs has contributed to the evolution of the human neocortex and plays an important role during human brain development.

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells.

The human brain is arguably the most complex structure among living organisms. However, the specific mechanisms leading to this complexity remain incompletely understood, primarily because of the poor experimental accessibility of the human embryonic brain. Over recent years, technologies based on pluripotent stem cells (PSCs) have been developed to generate neural cells of various types. While the translational potential of PSC technologies for disease modeling and/or cell replacement therapies is usually put forward as a rationale for their utility, they are also opening novel windows for direct observation and experimentation of the basic mechanisms of human brain development. PSC-based studies have revealed that a number of cardinal features of neural ontogenesis are remarkably conserved in human models, which can be studied in a reductionist fashion. They have also revealed species-specific features, which constitute attractive lines of investigation to elucidate the mechanisms underlying the development of the human brain, and its link with evolution.

tRNA methyltransferase homolog gene TRMT10A mutation in young onset diabetes and primary microcephaly in humans.

We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene TRMT10A (also called RG9MTD2). TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m(1)G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of ß- and non-ß-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human ß-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects ß-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin.

The transcription factor Eomesodermin (Eomes) is involved in early embryonic patterning, but the range of cell fates that it controls as well as its mechanisms of action remain unclear. Here we show that transient expression of Eomes promotes cardiovascular fate during embryonic stem cell differentiation. Eomes also rapidly induces the expression of Mesp1, a key regulator of cardiovascular differentiation, and directly binds to regulatory sequences of Mesp1. Eomes effects are strikingly modulated by Activin signalling: high levels of Activin inhibit the promotion of cardiac mesoderm by Eomes, while they enhance Eomes-dependent endodermal specification. These results place Eomes upstream of the Mesp1-dependent programme of cardiogenesis, and at the intersection of mesodermal and endodermal specification, depending on the levels of Activin/Nodal signalling.

An intrinsic mechanism of corticogenesis from embryonic stem cells.

The cerebral cortex develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved remain unclear. Here we show that mouse embryonic stem cells, cultured without any morphogen but in the presence of a sonic hedgehog inhibitor, recapitulate in vitro the major milestones of cortical development, leading to the sequential generation of a diverse repertoire of neurons that display most salient features of genuine cortical pyramidal neurons. When grafted into the cerebral cortex, these neurons develop patterns of axonal projections corresponding to a wide range of cortical layers, but also to highly specific cortical areas, in particular visual and limbic areas, thereby demonstrating that the identity of a cortical area can be specified without any influence from the brain. The discovery of intrinsic corticogenesis sheds new light on the mechanisms of neuronal specification, and opens new avenues for the modelling and treatment of brain diseases.

Metabolic mechanisms of species-specific developmental tempo.

Development consists of a highly ordered suite of steps and transitions, like choreography. Although these sequences are often evolutionarily conserved, they can display species variations in duration and speed, thereby modifying final organ size or function. Despite their evolutionary significance, the mechanisms underlying species-specific scaling of developmental tempo have remained unclear. Here, we will review recent findings that implicate global cellular mechanisms, particularly intermediary and protein metabolism, as species-specific modifiers of developmental tempo. In various systems, from somitic cell oscillations to neuronal development, metabolic pathways display species differences. These have been linked to mitochondrial metabolism, which can influence the species-specific speed of developmental transitions. Thus, intermediary metabolic pathways regulate developmental tempo together with other global processes, including proteostasis and chromatin remodeling. By linking metabolism and the evolution of developmental trajectories, these findings provide opportunities to decipher how species-specific cellular timing can influence organism fitness.

Linking mitochondria metabolism, developmental timing, and human brain evolution.

Changes in developmental timing are an important factor of evolution in organ shape and function. This is particularly striking for human brain development, which, compared with other mammals, is considerably prolonged at the level of the cerebral cortex, resulting in brain neoteny. Here, we review recent findings that indicate that mitochondria and metabolism contribute to species differences in the tempo of cortical neuron development. Mitochondria display species-specific developmental timeline and metabolic activity patterns that are highly correlated with the speed of neuron maturation. Enhancing mitochondrial activity in human cortical neurons results in their accelerated maturation, while its reduction leads to decreased maturation rates in mouse neurons. Together with other global and gene-specific mechanisms, mitochondria thus act as a cellular hourglass of neuronal developmental tempo and may thereby contribute to species-specific features of human brain ontogeny.

If you please, draw me a neuron - linking evolutionary tinkering with human neuron evolution.

Animal speciation often involves novel behavioral features that rely on nervous system evolution. Human-specific brain features have been proposed to underlie specialized cognitive functions and to be linked, at least in part, to the evolution of synapses, neurons, and circuits of the cerebral cortex. Here, we review recent results showing that, while the human cortex is composed of a repertoire of cells that appears to be largely similar to the one found in other mammals, human cortical neurons do display specialized features at many levels, from gene expression to intrinsic physiological properties. The molecular mechanisms underlying human species-specific neuronal features remain largely unknown but implicate hominid-specific gene duplicates that encode novel molecular modifiers of neuronal function. The identification of human-specific genetic modifiers of neuronal function brings novel insights on brain evolution and function and, could also provide new insights on human species-specific vulnerabilities to brain disorders.

Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons.

Neuronal activity-dependent transcription plays a key role in plasticity and pathology in the brain. An intriguing question is how neuronal activity controls gene expression via interactions of transcription factors with DNA and chromatin modifiers in the nucleus. By utilizing single-molecule imaging in human embryonic stem cell (ESC)-derived cortical neurons, we demonstrate that neuronal activity increases repetitive emergence of cAMP response element-binding protein (CREB) at histone acetylation sites in the nucleus, where RNA polymerase II (RNAPII) accumulation and FOS expression occur rapidly. Neuronal activity also enhances co-localization of CREB and CREB-binding protein (CBP). Increased binding of a constitutively active CREB to CBP efficiently induces CREB repetitive emergence. On the other hand, the formation of histone acetylation sites is dependent on CBP histone modification via acetyltransferase (HAT) activity but is not affected by neuronal activity. Taken together, our results suggest that neuronal activity promotes repetitive CREB-CRE and CREB-CBP interactions at predetermined histone acetylation sites, leading to rapid gene expression.

SYNGAP1 deficiency disrupts synaptic neoteny in xenotransplanted human cortical neurons in vivo.

Human brain ontogeny is characterized by a considerably prolonged neotenic development of cortical neurons and circuits. Neoteny is thought to be essential for the acquisition of advanced cognitive functions, which are typically altered in intellectual disability (ID) and autism spectrum disorders (ASDs). Human neuronal neoteny could be disrupted in some forms of ID and/or ASDs, but this has never been tested. Here, we use xenotransplantation of human cortical neurons into the mouse brain to model SYNGAP1 haploinsufficiency, one of the most prevalent genetic causes of ID/ASDs. We find that SYNGAP1-deficient human neurons display strong acceleration of morphological and functional synaptic formation and maturation alongside disrupted synaptic plasticity. At the circuit level, SYNGAP1-haploinsufficient neurons display precocious acquisition of responsiveness to visual stimulation months ahead of time. Our findings indicate that SYNGAP1 is required cell autonomously for human neuronal neoteny, providing novel links between human-specific developmental mechanisms and ID/ASDs.

Protocol to process fresh human cerebral cortex biopsies for patch-clamp recording and immunostaining.

Cerebral cortex biopsies enable the investigation of native developing and mature human brain tissue. Here, we present a protocol to process human cortical biopsies from the surgical theater to the laboratory. We describe steps for the preparation of viable acute slices for patch-clamp recording using dedicated chemical solutions for transport and sectioning. We then explain procedures for tissue fixation and post hoc immunostaining to correlate physiological properties to morphological features and protein detection. For complete details on the use and execution of this protocol, please refer to Libé-Philippot et al.1.

CTNND2 moderates the pace of synaptic maturation and links human evolution to synaptic neoteny.

Human-specific genes are potential drivers of brain evolution. Among them, SRGAP2C has contributed to the emergence of features characterizing human cortical synapses, including their extended period of maturation. SRGAP2C inhibits its ancestral copy, the postsynaptic protein SRGAP2A, but the synaptic molecular pathways differentially regulated in humans by SRGAP2 proteins remain largely unknown. Here, we identify CTNND2, a protein implicated in severe intellectual disability (ID) in Cri-du-Chat syndrome, as a major partner of SRGAP2. We demonstrate that CTNND2 slows synaptic maturation and promotes neuronal integrity. During postnatal development, CTNND2 moderates neuronal excitation and excitability. In adults, it supports synapse maintenance. While CTNND2 deficiency is deleterious and results in synaptic loss of SYNGAP1, another major ID-associated protein, the human-specific protein SRGAP2C, enhances CTNND2 synaptic accumulation in human neurons. Our findings suggest that CTNND2 regulation by SRGAP2C contributes to synaptic neoteny in humans and link human-specific and ID genes at the synapse.

Synaptic neoteny of human cortical neurons requires species-specific balancing of SRGAP2-SYNGAP1 cross-inhibition.

Human-specific (HS) genes have been implicated in brain evolution, but their impact on human neuron development and diseases remains unclear. Here, we study SRGAP2B/C, two HS gene duplications of the ancestral synaptic gene SRGAP2A, in human cortical pyramidal neurons (CPNs) xenotransplanted in the mouse cortex. Downregulation of SRGAP2B/C in human CPNs led to strongly accelerated synaptic development, indicating their requirement for the neoteny that distinguishes human synaptogenesis. SRGAP2B/C genes promoted neoteny by reducing the synaptic levels of SRGAP2A,thereby increasing the postsynaptic accumulation of the SYNGAP1 protein, encoded by a major intellectual disability/autism spectrum disorder (ID/ASD) gene. Combinatorial loss-of-function experiments in vivo revealed that the tempo of synaptogenesis is set by the reciprocal antagonism between SRGAP2A and SYNGAP1, which in human CPNs is tipped toward neoteny by SRGAP2B/C. Thus, HS genes can modify the phenotypic expression of genetic mutations leading to ID/ASD through the regulation of human synaptic neoteny.

Transcriptional mechanisms of EphA7 gene expression in the developing cerebral cortex.

The patterning of cortical areas is controlled by a combination of intrinsic factors that are expressed in the cortex and external signals such as inputs from the thalamus. EphA7 is a guidance receptor that is involved in key aspects of cortical development and is expressed in gradients within developing cortical areas. Here, we identified a regulatory element of the EphA7 promoter, named pA7, that can recapitulate salient features of the pattern of expression of EphA7, including cortical gradients. Using a pA7-Green fluorescent Protein (GFP) mouse reporter line, we isolated cortical neuron populations displaying different levels of EphA7/GFP expression. Transcriptome analysis of these populations enabled to identify many differentially expressed genes, including 26 transcription factors with putative binding sites in the pA7 element. Among these, Pbx1 was found to bind directly to the EphA7 promoter in the developing cortex. All genes validated further were confirmed to be expressed differentially in the developing cortex, similarly to EphA7. Their expression was unchanged in mutant mice defective for thalamocortical projections, indicating a transcriptional control largely intrinsic to the cortex. Our study identifies a novel repertoire of cortical neuron genes that may act upstream of, or together with EphA7, to control the patterning of cortical areas.