Mechanisms Underlying Circuit Dysfunction in Neurodevelopmental Disorders.

Recent advances in genomics have revealed a wide spectrum of genetic variants associated with neurodevelopmental disorders at an unprecedented scale. An increasing number of studies have consistently identified mutations-both inherited and de novo-impacting the function of specific brain circuits. This suggests that, during brain development, alterations in distinct neural circuits, cell types, or broad regulatory pathways ultimately shaping synapses might be a dysfunctional process underlying these disorders. Here, we review findings from human studies and animal model research to provide a comprehensive description of synaptic and circuit mechanisms implicated in neurodevelopmental disorders. We discuss how specific synaptic connections might be commonly disrupted in different disorders and the alterations in cognition and behaviors emerging from imbalances in neuronal circuits. Moreover, we review new approaches that have been shown to restore or mitigate dysfunctional processes during specific critical windows of brain development. Considering the heterogeneity of neurodevelopmental disorders, we also highlight the recent progress in developing improved clinical biomarkers and strategies that will help to identify novel therapeutic compounds and opportunities for early intervention.

Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling.

Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (gamma-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

Focal adhesion kinase regulates actin nucleation and neuronal filopodia formation during axonal growth.

The establishment of neural circuits depends on the ability of axonal growth cones to sense their surrounding environment en route to their target. To achieve this, a coordinated rearrangement of cytoskeleton in response to extracellular cues is essential. Although previous studies have identified different chemotropic and adhesion molecules that influence axonal development, the molecular mechanism by which these signals control the cytoskeleton remains poorly understood. Here, we show that in vivo conditional ablation of the focal adhesion kinase gene (Fak) from mouse hippocampal pyramidal cells impairs axon outgrowth and growth cone morphology during development, which leads to functional defects in neuronal connectivity. Time-lapse recordings and in vitro FRAP analysis indicate that filopodia motility is altered in growth cones lacking FAK, probably owing to deficient actin turnover. We reveal the intracellular pathway that underlies this process and describe how phosphorylation of the actin nucleation-promoting factor N-WASP is required for FAK-dependent filopodia formation. Our study reveals a novel mechanism through which FAK controls filopodia formation and actin nucleation during axonal development.

Focal adhesion kinase modulates radial glia-dependent neuronal migration through connexin-26.

Focal adhesion kinase (FAK) is an intracellular kinase and scaffold protein that regulates migration in many different cellular contexts but whose function in neuronal migration remains controversial. Here, we have analyzed the function of FAK in two populations of neurons with very distinct migratory behaviors: cortical interneurons, which migrate tangentially and independently of radial glia; and pyramidal cells, which undergo glial-dependent migration. We found that FAK is dispensable for glial-independent migration but is cell-autonomously required for the normal interaction of pyramidal cells with radial glial fibers. Loss of FAK function disrupts the normal morphology of migrating pyramidal cells, delays migration, and increases the tangential dispersion of neurons arising from the same radial unit. FAK mediates this process by regulating the assembly of Connexin-26 contact points in the membrane of migrating pyramidal cells. These results indicate that FAK plays a fundamental role in the dynamic regulation of Gap-mediated adhesions during glial-guided neuronal migration in the mouse.

CREB-Dependent Regulation of GAD65 Transcription by BDNF/TrkB in Cortical Interneurons.

In the cerebral cortex, the functional output of projection neurons is fine-tuned by inhibitory neurons present in the network, which use γ-aminobutyric acid (GABA) as their main neurotransmitter. Previous studies have suggested that the expression levels of the rate-limiting GABA synthetic enzyme, GAD65, depend on brain derived neurotrophic factor (BDNF)/TrkB activation. However, the molecular mechanisms by which this neurotrophic factor and its receptor controls GABA synthesis are still unknown. Here, we show a direct regulation of the GAD65 gene by BDNF-TrkB signaling via CREB in cortical interneurons. Conditional ablation of TrkB in cortical interneurons causes a cell-autonomous decrease in the synaptically enriched GAD65 protein and its transcripts levels, suggesting that transcriptional regulation of the GAD65 gene is altered. Dissection of the intracellular pathway that underlies this process revealed that BDNF/TrkB signaling controls the transcription of GAD65 in a Ras-ERK-CREB-dependent manner. Our study reveals a novel molecular mechanism through which BDNF/TrkB signaling may modulate the maturation and function of cortical inhibitory circuits.

Dissecting cellular diversity of cortical GABAergic cells across multiple modalities: A turning point in neuronal taxonomy.

Decoding the complexity of the brain requires an understanding of the architecture, function, and development of its neuronal circuits. Neuronal classifications that group neurons based on specific features/behaviors have become essential to further analyze the different subtypes in a systematic and reproducible way. A comprehensive taxonomic framework, accounting for multiple defining and quantitative features, will provide the reference to infer generalized rules for cells ascribed to the same neuronal type, and eventually predict cellular behaviors, even in the absence of experimental measures. Technologies that enable cell-type classification in the nervous system are rapidly evolving in scalability and resolution. While these approaches depict astonishing diversity in neuronal morphology, electrophysiology, and gene expression, a robust metric of the coherence between different profiling modalities leading to a unified classification is still largely missing. Focusing on GABAergic neurons of the cerebral cortex, Gouwens et al.1 pioneered the first integrated cell-type classification based on the simultaneous analysis of the transcriptional networks, the recording of intrinsic electrophysiological properties, and the reconstruction of 3D morphologies of the same cell. Their comprehensive and high-quality data provide a new framework to shed light on what may be considered a "neuronal cell type."

Cortical wiring by synapse type-specific control of local protein synthesis.

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.

BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-beta-catenin interactions.

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles "splitting" away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin-beta-catenin adhesion complexes that occurs after tyrosine phosphorylation of beta-catenin. Artificially maintaining cadherin-beta-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin-beta-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

Focal adhesion kinase functions downstream of Sema3A signaling during axonal remodeling.

Axon refinement is a necessary event for sculpting the final wiring of neural circuits. Although some factors have been identified that cause axonal arbor remodeling, the molecular pathways transducing these extracellular signals to adhesion disassembly and the cytoskeleton are poorly understood. Here we show that conditional ablation of Focal adhesion kinase (Fak) abolishes axon remodeling induced by Semaphorin-3A (Sema3A) in hippocampal neurons. Sema3A elicits divergent effects on different tyrosine residues of FAK: it increases phosphorylation of Tyr397, the kinase domain and the tyrosine residue 925, and decreases phosphorylation of Tyr407 and Tyr861. Moreover, Sema3A mediates mechanisms that contribute to the disassembly of adhesion contacts in a FAK-dependent manner: tyrosine phosphorylation of alpha-actinin and FAKY925 that decreases FAK-Paxillin interaction. Altogether, our results provide novel insights into the spatiotemporal dynamics of FAK activation mediated by Sema3A and on its interaction with its downstream effectors: Paxillin and alpha-actinin in neurons.

Subcellular sorting of neuregulins controls the assembly of excitatory-inhibitory cortical circuits.

The assembly of specific neuronal circuits relies on the expression of complementary molecular programs in presynaptic and postsynaptic neurons. In the cerebral cortex, the tyrosine kinase receptor ErbB4 is critical for the wiring of specific populations of GABAergic interneurons, in which it paradoxically regulates both the formation of inhibitory synapses as well as the development of excitatory synapses received by these cells. Here, we found that Nrg1 and Nrg3, two members of the neuregulin family of trophic factors, regulate the inhibitory outputs and excitatory inputs of interneurons in the mouse cerebral cortex, respectively. The differential role of Nrg1 and Nrg3 in this process is not due to their receptor-binding EGF-like domain, but rather to their distinctive subcellular localization within pyramidal cells. Our study reveals a novel strategy for the assembly of cortical circuits that involves the differential subcellular sorting of family-related synaptic proteins.

Distinct molecular programs regulate synapse specificity in cortical inhibitory circuits.

How neuronal connections are established and organized into functional networks determines brain function. In the mammalian cerebral cortex, different classes of GABAergic interneurons exhibit specific connectivity patterns that underlie their ability to shape temporal dynamics and information processing. Much progress has been made toward parsing interneuron diversity, yet the molecular mechanisms by which interneuron-specific connectivity motifs emerge remain unclear. In this study, we investigated transcriptional dynamics in different classes of interneurons during the formation of cortical inhibitory circuits in mouse. We found that whether interneurons form synapses on the dendrites, soma, or axon initial segment of pyramidal cells is determined by synaptic molecules that are expressed in a subtype-specific manner. Thus, cell-specific molecular programs that unfold during early postnatal development underlie the connectivity patterns of cortical interneurons.

TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum.

Neurotrophins are essential to the normal development and maintenance of the nervous system. Neurotrophin signaling is mediated by Trk family tyrosine kinases such as TrkA, TrkB and TrkC, as well as by the pan-neurotrophin receptor p75NTR. Here we have deleted the trkB gene in cerebellar precursors by Wnt1-driven Cre--mediated recombination to study the function of the TrkB in the cerebellum. Despite the absence of TrkB, the mature cerebellum of mutant mice appears similar to that of wild type, with all types of cell present in normal numbers and positions. Granule and Purkinje cell dendrites appear normal and the former have typical numbers of excitatory synapses. By contrast, inhibitory interneurons are strongly affected: although present in normal numbers, they express reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations. Thus, TrkB is essential to the development of GABAergic neurons and regulates synapse formation in addition to its role in the development of axon terminals.

Control of axonal branching and synapse formation by focal adhesion kinase.

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.

Molecular diversity underlying cortical excitatory and inhibitory synapse development.

The complexity and precision of cortical circuitries is achieved during development due to the exquisite diversity of synapse types that is generated in a highly regulated manner. Here, we review the recent increase in our understanding of how synapse type-specific molecules differentially regulate the development of excitatory and inhibitory synapses. Moreover, several synapse subtype-specific molecules have been shown to control the targeting, formation or maturation of particular subtypes of excitatory synapses. Because inhibitory neurons are extremely diverse, a similar molecular diversity is likely to underlie the development of different inhibitory synapses making it a promising topic for future investigation in the field of the synapse development.

The Microtubule Regulator NEK7 Coordinates the Wiring of Cortical Parvalbumin Interneurons.

Functional networks in the mammalian cerebral cortex rely on the interaction between glutamatergic pyramidal cells and GABAergic interneurons. Both neuronal populations exhibit an extraordinary divergence in morphology and targeting areas, which ultimately dictate their precise function in cortical circuits. How these prominent morphological differences arise during development is not well understood. Here, we conducted a high-throughput screen for genes differentially expressed by pyramidal cells and interneurons during cortical wiring. We found that NEK7, a kinase involved in microtubule polymerization, is mostly expressed in parvalbumin (PV+) interneurons at the time when they establish their connectivity. Functional experiments revealed that NEK7-deficient PV+ interneurons show altered microtubule dynamics, axon growth cone steering and reduced axon length, arbor complexity, and total number of synaptic contacts formed with pyramidal cells. Altogether, our results reveal a molecular mechanism by which the microtubule-associated kinase NEK7 regulates the wiring of PV+ interneurons.

Neural circuit dysfunction in mouse models of neurodevelopmental disorders.

Neuropsychiatric disorders arise from the alteration of normal brain developmental trajectories disrupting the function of specific neuronal circuits. Recent advances in human genetics have greatly accelerated the identification of genes whose variation increases the susceptibility for neurodevelopmental disorders, most notably for autism spectrum disorder (ASD) and schizophrenia. In parallel, experimental studies in animal models-most typically in mice-are beginning to shed light on the role of these genes in the development and function of specific brain circuits. In spite of their limitations, understanding the impact of pathological gene variation in animal models at the level of specific neuronal populations and circuits will likely contribute to orienting human clinical studies in the search for precise disease mechanisms and novel treatments.

Critical role of integrin-linked kinase in granule cell precursor proliferation and cerebellar development.

Integrin-linked kinase (ILK) is a serine/threonine protein kinase that plays an important role in integrin signaling and cell proliferation. We used Cre recombinase (Cre)-loxP technology to study CNS restricted knock-out of the ilk gene by either Nestin-driven or gfap-driven Cre-mediated recombination. Developmental changes in ilk-excised brain regions are similar to those observed in mice lacking the integrin beta1 subunit in the CNS, including defective laminin deposition, abnormal glial morphology, and alterations in granule cell migration. Decreases in 6-bromodeoxyuridine (BrdU) pulse labeling and proliferating cell nuclear antigen expression in the external granule cell layer of the cerebellum demonstrated that proliferation is disrupted in granule cells lacking ILK. Previous studies have shown that laminin-sonic hedgehog (Shh)-induced granule cell precursor (GCP) proliferation is dependent on beta1 integrins, several of which bind laminin and interact with ILK through the beta1 cytoplasmic domain. Both ex vivo deletion of ilk and a small molecule inhibitor of ILK kinase activity decreased laminin-Shh-induced BrdU labeling in cultured GCPs. Together, these results implicate ILK as a critical effector in a signaling pathway necessary for granule cell proliferation and cerebellar development.

Abnormal wiring of CCK+ basket cells disrupts spatial information coding.

The function of cortical GABAergic interneurons is largely determined by their integration into specific neural circuits, but the mechanisms controlling the wiring of these cells remain largely unknown. This is particularly true for a major population of basket cells that express the neuropeptide cholecystokinin (CCK). Here we found that the tyrosine kinase receptor ErbB4 was required for the normal integration into cortical circuits of basket cells expressing CCK and vesicular glutamate transporter 3 (VGlut3). The number of inhibitory synapses made by CCK+VGlut3+ basket cells and the inhibitory drive they exerted on pyramidal cells were reduced in conditional mice lacking ErbB4. Developmental disruption of the connectivity of these cells diminished the power of theta oscillations during exploratory behavior, disrupted spatial coding by place cells, and caused selective alterations in spatial learning and memory in adult mice. These results suggest that normal integration of CCK+ basket cells in cortical networks is key to support spatial coding in the hippocampus.

Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican.

Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.