Nova proteins direct synaptic integration of somatostatin interneurons through activity-dependent alternative splicing.

Somatostatin interneurons are the earliest born population of cortical inhibitory cells. They are crucial to support normal brain development and function; however, the mechanisms underlying their integration into nascent cortical circuitry are not well understood. In this study, we begin by demonstrating that the maturation of somatostatin interneurons in mouse somatosensory cortex is activity dependent. We then investigated the relationship between activity, alternative splicing, and synapse formation within this population. Specifically, we discovered that the Nova family of RNA-binding proteins are activity-dependent and are essential for the maturation of somatostatin interneurons, as well as their afferent and efferent connectivity. Within this population, Nova2 preferentially mediates the alternative splicing of genes required for axonal formation and synaptic function independently from its effect on gene expression. Hence, our work demonstrates that the Nova family of proteins through alternative splicing are centrally involved in coupling developmental neuronal activity to cortical circuit formation.

The brain's polymath: Emerging roles of microglia throughout brain development.

Microglia, the resident brain immune cells, have garnered a reputation as major effectors of circuit wiring due to their ability to prune synapses. Other roles of microglia in regulating neuronal circuit development have so far received comparatively less attention. Here, we review the latest studies that have contributed to our increased understanding of how microglia regulate brain wiring beyond their role in synapse pruning. We summarize recent findings showing that microglia regulate neuronal numbers and influence neuronal connectivity through a bidirectional communication between microglia and neurons, processes regulated by neuronal activity and the remodeling of the extracellular matrix. Finally, we speculate on the potential contribution of microglia to the development of functional networks and propose an integrative view of microglia as active elements of neural circuits.

Microglia contribute to the postnatal development of cortical somatostatin-positive inhibitory cells and to whisker-evoked cortical activity.

Microglia play a key role in shaping the formation and refinement of the excitatory network of the brain. However, less is known about whether and how they organize the development of distinct inhibitory networks. We find that microglia are essential for the proper development of somatostatin-positive (SST+) cell synapses during the second postnatal week. We further identify a pair of molecules that act antagonistically to one another in the organization of SST+ cell axonal elaboration. Whereas CX3CL1 acts to suppress axonal growth and complexity, CXCL12 promotes it. Assessing the functional importance of microglia in the development of cortical activity, we find that a whisker stimulation paradigm that drives SST+ cell activation leads to reduced cortical spiking in brains depleted of microglia. Collectively, our data demonstrate an important role of microglia in regulating the development of SST+ cell output early in life.

Monitoring phagocytic uptake of amyloid ß into glial cell lysosomes in real time.

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer's disease (AD) have primarily focused on targeting antibodies to amyloid ß (Aß) or inhibitng enzymes that make it, and while removal of Aß by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aß1-42 analogue (AßpH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of AßpH in real time in live animals. We find that microglia phagocytose more AßpH than astrocytes in culture, in brain slices and in vivo. AßpH can be used to investigate the phagocytic mechanisms responsible for removing Aß from the extracellular space, and thus could become a useful tool to study Aß clearance at different stages of AD.

GABA-receptive microglia selectively sculpt developing inhibitory circuits.

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.

Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

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.

Rbfox1 Mediates Cell-type-Specific Splicing in Cortical Interneurons.

Cortical interneurons display a remarkable diversity in their morphology, physiological properties, and connectivity. Elucidating the molecular determinants underlying this heterogeneity is essential for understanding interneuron development and function. We discovered that alternative splicing differentially regulates the integration of somatostatin- and parvalbumin-expressing interneurons into nascent cortical circuits through the cell-type-specific tailoring of mRNAs. Specifically, we identified a role for the activity-dependent splicing regulator Rbfox1 in the development of cortical interneuron-subtype-specific efferent connectivity. Our work demonstrates that Rbfox1 mediates largely non-overlapping alternative splicing programs within two distinct but related classes of interneurons.

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.

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.

Shaping Early Networks to Rule Mature Circuits: Little MiRs Go a Long Way.

Normative cortical processing depends on precise interactions between excitatory and inhibitory neurons. In this issue of Neuron, Lippi et al. (2016) identify miR-101 as a master regulator coordinating molecular programs during development that ultimately impact the activity of mature networks.

Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis.

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

The action mechanism of the Myc inhibitor termed Omomyc may give clues on how to target Myc for cancer therapy.

Recent evidence points to Myc--a multifaceted bHLHZip transcription factor deregulated in the majority of human cancers--as a priority target for therapy. How to target Myc is less clear, given its involvement in a variety of key functions in healthy cells. Here we report on the action mechanism of the Myc interfering molecule termed Omomyc, which demonstrated astounding therapeutic efficacy in transgenic mouse cancer models in vivo. Omomyc action is different from the one that can be obtained by gene knockout or RNA interference, approaches designed to block all functions of a gene product. This molecule--instead--appears to cause an edge-specific perturbation that destroys some protein interactions of the Myc node and keeps others intact, with the result of reshaping the Myc transcriptome. Omomyc selectively targets Myc protein interactions: it binds c- and N-Myc, Max and Miz-1, but does not bind Mad or select HLH proteins. Specifically, it prevents Myc binding to promoter E-boxes and transactivation of target genes while retaining Miz-1 dependent binding to promoters and transrepression. This is accompanied by broad epigenetic changes such as decreased acetylation and increased methylation at H3 lysine 9. In the presence of Omomyc, the Myc interactome is channeled to repression and its activity appears to switch from a pro-oncogenic to a tumor suppressive one. Given the extraordinary therapeutic impact of Omomyc in animal models, these data suggest that successfully targeting Myc for cancer therapy might require a similar twofold action, in order to prevent Myc/Max binding to E-boxes and, at the same time, keep repressing genes that would be repressed by Myc.