Postnatal TrkB ablation in corticolimbic interneurons induces social dominance in male mice.

The tight balance between synaptic excitation and inhibition (E/I) within neocortical circuits in the mammalian brain is important for complex behavior. Many loss-of-function studies have demonstrated that brain-derived neurotrophic factor (BDNF) and its cognate receptor tropomyosin receptor kinase B (TrkB) are essential for the development of inhibitory GABAergic neurons. However, behavioral consequences of impaired BDNF/TrkB signaling in GABAergic neurons remain unclear, largely due to confounding motor function deficits observed in previous animal models. In this study, we generated conditional knockout mice (TrkB cKO) in which TrkB was ablated from a majority of corticolimbic GABAergic interneurons postnatally. These mice showed intact motor coordination and movement, but exhibited enhanced dominance over other mice in a group-housed setting. In addition, immature fast-spiking GABAergic neurons of TrkB cKO mice resulted in an E/I imbalance in layer 5 microcircuits within the medial prefrontal cortex (mPFC), a key region regulating social dominance. Restoring the E/I imbalance via optogenetic modulation in the mPFC of TrkB cKO mice normalized their social dominance behavior. Taken together, our results provide strong evidence for a role of BDNF/TrkB signaling in inhibitory synaptic modulation and social dominance behavior in mice.

Visualizing Microglia with a Fluorescence Turn-On Ugt1a7c Substrate.

Microglia, the brain-resident macrophage, are involved in brain development and contribute to the progression of neural disorders. Despite the importance of microglia, imaging of live microglia at a cellular resolution has been limited to transgenic mice. Efforts have therefore been dedicated to developing new methods for microglia detection and imaging. Using a thorough structure-activity relationships study, we developed CDr20, a high-performance fluorogenic chemical probe that enables the visualization of microglia both in vitro and in vivo. Using a genome-scale CRISPR-Cas9 knockout screen, the UDP-glucuronosyltransferase Ugt1a7c was identified as the target of CDr20. The glucuronidation of CDr20 by Ugt1a7c in microglia produces fluorescence.

MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development.

The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2-dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

Role of pro-brain-derived neurotrophic factor (proBDNF) to mature BDNF conversion in activity-dependent competition at developing neuromuscular synapses.

Formation of specific neuronal connections often involves competition between adjacent axons, leading to stabilization of the active terminal, while retraction of the less active ones. The underlying molecular mechanisms remain unknown. We show that activity-dependent conversion of pro-brain-derived neurotrophic factor (proBDNF) to mature (m)BDNF mediates synaptic competition. Stimulation of motoneurons triggers proteolytic conversion of proBDNF to mBDNF at nerve terminals. In Xenopus nerve-muscle cocultures, in which two motoneurons innervate one myocyte, proBDNF-p75(NTR) signaling promotes retraction of the less active terminal, whereas mBDNF-tyrosine-related kinase B (TrkB) p75NTR (p75 neurotrophin receptor) facilitates stabilization of the active one. Thus, proBDNF and mBDNF may serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, and activity-dependent conversion of proBDNF to mBDNF may regulate synapse elimination.

ProBDNF and mature BDNF as punishment and reward signals for synapse elimination at mouse neuromuscular junctions.

During development, mammalian neuromuscular junctions (NMJs) transit from multiple-innervation to single-innervation through axonal competition via unknown molecular mechanisms. Previously, using an in vitro model system, we demonstrated that the postsynaptic secretion of pro-brain-derived neurotrophic factor (proBDNF) stabilizes or eliminates presynaptic axon terminals, depending on its proteolytic conversion at synapses. Here, using developing mouse NMJs, we obtained in vivo evidence that proBDNF and mature BDNF (mBDNF) play roles in synapse elimination. We observed that exogenous proBDNF promoted synapse elimination, whereas mBDNF infusion substantially delayed synapse elimination. In addition, pharmacological inhibition of the proteolytic conversion of proBDNF to mBDNF accelerated synapse elimination via activation of p75 neurotrophin receptor (p75(NTR)). Furthermore, the inhibition of both p75(NTR) and sortilin signaling attenuated synapse elimination. We propose a model in which proBDNF and mBDNF serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, in vivo.

Human pluripotent stem cell (hPSC)-derived microglia for the study of brain disorders. A comprehensive review of existing protocols.

Microglia, resident immune cells of the brain that originate from the yolk sac, play a critical role in maintaining brain homeostasis by monitoring and phagocytosing pathogens and cellular debris in the central nervous system (CNS). While they share characteristics with myeloid cells, they are distinct from macrophages. In response to injury, microglia release pro-inflammatory factors and contribute to brain homeostasis through activities such as synapse pruning and neurogenesis. To better understand their role in neurological disorders, the generation of in vitro models of human microglia has become essential. These models, derived from patient-specific induced pluripotent stem cells (iPSCs), provide a controlled environment to study the molecular and cellular mechanisms underlying microglia-mediated neuroinflammation and neurodegeneration. The incorporation or generation of microglia into three-dimensional (3D) organoid cultures provides a more physiologically relevant environment that offers further opportunities to study microglial dynamics and disease modeling. This review describes several protocols that have been recently developed for the generation of human-induced microglia. Importantly, it highlights the promise of these in vitro models in advancing our understanding of brain disorders and facilitating personalized drug screening.

Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3.

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

Prefrontal cortical mechanisms underlying delayed alternation in mice.

The prefrontal cortex (PFC) has been implicated in the maintenance of task-relevant information during goal-directed behavior. Using a combination of lesions, local inactivation, and optogenetics, we investigated the functional role of the medial prefrontal cortex (mPFC) in mice with a novel operant delayed alternation task. Task difficulty was manipulated by changing the duration of the delay between two sequential actions. In experiment 1, we showed that excitotoxic lesions of the mPFC impaired acquisition of delayed alternation with long delays (16 s), whereas lesions of the dorsal hippocampus and ventral striatum, areas connected with the PFC, did not produce any deficits. Lesions of dorsal hippocampus, however, significantly impaired reversal learning when the rule was changed from alternation to repetition. In experiment 2, we showed that local infusions of muscimol (an agonist of the GABA(A) receptor) into mPFC impaired performance even when the animal was well trained, suggesting that the mPFC is critical not only for acquisition but also for successful performance. In experiment 3, to examine the mechanisms underlying the role of GABAergic inhibition, we used Cre-inducible Channelrhodopsin-2 to activate parvalbumin (PV)-expressing GABAergic interneurons in the mPFC of PV-Cre transgenic mice as they performed the task. Using whole cell patch-clamp recording, we demonstrated that activation of PV-expressing interneurons in vitro with blue light in brain slices reliably produced spiking and inhibited nearby pyramidal projection neurons. With similar stimulation parameters, in vivo stimulation significantly impaired delayed alternation performance. Together these results demonstrate a critical role for the mPFC in the acquisition and performance of the delayed alternation task.

Neural circuit analysis using a novel intersectional split intein-mediated split-Cre recombinase system.

The defining features of a neuron are its functional and anatomical connections with thousands of other neurons in the brain. Together, these neurons form functional networks that direct animal behavior. Current approaches that allow the interrogation of specific populations of neurons and neural circuits rely heavily on targeting their gene expression profiles or connectivity. However, these approaches are often unable to delineate specific neuronal populations. Here, we developed a novel intersectional split intein-mediated split-Cre recombinase system that can selectively label specific types of neurons based on their gene expression profiles and structural connectivity. We developed this system by splitting Cre recombinase into two fragments with evolved split inteins and subsequently expressed one fragment under the influence of a cell type-specific promoter in a transgenic animal, and delivered the other fragment via retrograde viral gene transfer. This approach results in the reconstitution of Cre recombinase in only specific population of neurons projecting from a specific brain region or in those of a specific neuronal type. Taken together, our split intein-based split-Cre system will be useful for sophisticated characterization of mammalian brain circuits.

Prenatal selective serotonin reuptake inhibitor (SSRI) exposure induces working memory and social recognition deficits by disrupting inhibitory synaptic networks in male mice.

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed antidepressant drugs in pregnant women. Infants born following prenatal exposure to SSRIs have a higher risk for behavioral abnormalities, however, the underlying mechanisms remains unknown. Therefore, we examined the effects of prenatal fluoxetine, the most commonly prescribed SSRI, in mice. Intriguingly, chronic in utero fluoxetine treatment impaired working memory and social novelty recognition in adult males. In the medial prefrontal cortex (mPFC), a key region regulating these behaviors, we found augmented spontaneous inhibitory synaptic transmission onto the layer 5 pyramidal neurons. Fast-spiking interneurons in mPFC exhibited enhanced intrinsic excitability and serotonin-induced excitability due to upregulated serotonin (5-HT) 2A receptor (5-HT2AR) signaling. More importantly, the behavioral deficits in prenatal fluoxetine treated mice were reversed by the application of a 5-HT2AR antagonist. Taken together, our findings suggest that alterations in inhibitory neuronal modulation are responsible for the behavioral alterations following prenatal exposure to SSRIs.

Ca2+-dependent demethylation of phosphatase PP2Ac promotes glucose deprivation-induced cell death independently of inhibiting glycolysis.

Cancer cells increase glucose metabolism to support aerobic glycolysis. However, only some cancer cells are acutely sensitive to glucose withdrawal, and the underlying mechanism of this selective sensitivity is unclear. We showed that glucose deprivation initiates a cell death pathway in cancer cells that is dependent on the kinase RIPK1. Glucose withdrawal triggered rapid plasma membrane depolarization and an influx of extracellular calcium into the cell through the L-type calcium channel Cav1.3 (CACNA1D), followed by activation of the kinase CAMK1. CAMK1 and the demethylase PPME1 were required for the subsequent demethylation and inactivation of the catalytic subunit of the phosphatase PP2A (PP2Ac) and the phosphorylation of RIPK1. Plasma membrane depolarization, PP2Ac demethylation, and cell death were prevented by glucose and, unexpectedly, by its nonmetabolizable analog 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor. These findings reveal a previously unknown function of glucose as a signaling molecule that protects cells from death induced by plasma membrane depolarization, independently of its role in glycolysis. Components of this cancer cell death pathway represent potential therapeutic targets against cancer.

Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons.

Regulation of Brain-Derived Neurotrophic Factor Exocytosis and Gamma-Aminobutyric Acidergic Interneuron Synapse by the Schizophrenia Susceptibility Gene Dysbindin-1.

BACKGROUND: Genetic variations in dystrobrevin binding protein 1 (DTNBP1 or dysbindin-1) have been implicated as risk factors in the pathogenesis of schizophrenia. The encoded protein dysbindin-1 functions in the regulation of synaptic activity and synapse development. Intriguingly, a loss of function mutation in Dtnbp1 in mice disrupted both glutamatergic and gamma-aminobutyric acidergic transmission in the cerebral cortex; pyramidal neurons displayed enhanced excitability due to reductions in inhibitory synaptic inputs. However, the mechanism by which reduced dysbindin-1 activity causes inhibitory synaptic deficits remains unknown. METHODS: We investigated the role of dysbindin-1 in the exocytosis of brain-derived neurotrophic factor (BDNF) from cortical excitatory neurons, organotypic brain slices, and acute slices from dysbindin-1 mutant mice and determined how this change in BDNF exocytosis transsynaptically affected the number of inhibitory synapses formed on excitatory neurons via whole-cell recordings, immunohistochemistry, and live-cell imaging using total internal reflection fluorescence microscopy. RESULTS: A decrease in dysbindin-1 reduces the exocytosis of BDNF from cortical excitatory neurons, and this reduction in BDNF exocytosis transsynaptically resulted in reduced inhibitory synapse numbers formed on excitatory neurons. Furthermore, application of exogenous BDNF rescued the inhibitory synaptic deficits caused by the reduced dysbindin-1 level in both cultured cortical neurons and slice cultures. CONCLUSIONS: Taken together, our results demonstrate that these two genes linked to risk for schizophrenia (BDNF and dysbindin-1) function together to regulate interneuron development and cortical network activity. This evidence supports the investigation of the association between dysbindin-1 and BDNF in humans with schizophrenia.

Amyloid precursor protein regulates neurogenesis by antagonizing miR-574-5p in the developing cerebral cortex.

Amyloid precursor protein (APP) is a transmembrane glycoprotein proteolytically processed to release amyloid beta, a pathological hallmark of Alzheimer's disease. APP is expressed throughout the developing and mature brain; however, the primary function of this protein is unknown. We previously demonstrated that APP deficiency enhances neurogenesis, but the mechanisms underlying this process are not known. Here we show that APP regulates the expression of microRNAs in the cortex and in neural progenitors, specifically repressing miR-574-5p. We also show that overexpression of miR-574-5p promotes neurogenesis, but reduces the neural progenitor pool. In contrast, the reduced expression of miR-574-5p inhibits neurogenesis and stimulates proliferation in vitro and in utero. We further demonstrate that the inhibition of miR-574-5p in APP-knockout mice rescues the phenotypes associated with APP deficiency in neurogenesis. Taken together, these results reveal a mechanism in which APP regulates the neurogenesis through miRNA-mediated post-transcriptional regulation.

Triad3A regulates synaptic strength by ubiquitination of Arc.

Activity-dependent gene transcription and protein synthesis underlie many forms of learning-related synaptic plasticity. At excitatory glutamatergic synapses, the immediate early gene product Arc/Arg3.1 couples synaptic activity to postsynaptic endocytosis of AMPA-type glutamate receptors. Although the mechanisms for Arc induction have been described, little is known regarding the molecular machinery that terminates Arc function. Here, we demonstrate that the RING domain ubiquitin ligase Triad3A/RNF216 ubiquitinates Arc, resulting in its rapid proteasomal degradation. Triad3A associates with Arc, localizes to clathrin-coated pits, and is associated with endocytic sites in dendrites and spines. In the absence of Triad3A, Arc accumulates, leading to the loss of surface AMPA receptors. Furthermore, loss of Triad3A mimics and occludes Arc-dependent forms of synaptic plasticity. Thus, degradation of Arc by clathrin-localized Triad3A regulates the availability of synaptic AMPA receptors and temporally tunes Arc-mediated plasticity at glutamatergic synapses.

Presynaptic protein synthesis required for NT-3-induced long-term synaptic modulation.

BACKGROUND: Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. RESULTS: Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR) is fused with bacterial gyrase B domain (GyrB-PKR), which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. CONCLUSIONS: Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner.