A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward.

Visualization of plasticity in fear-evoked calcium signals in midbrain dopamine neurons.

Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium indicator GCaMP3. We observed calcium transients in a subset of dopamine neurons to an unconditioned fear stimulus on the first day of Pavlovian fear conditioning. On the second day, calcium transients occurred in response to conditioned and unconditioned stimuli. These results demonstrate plasticity in dopamine neuron calcium signals and the occurrence of activity-dependent processes in these neurons during fear conditioning.

Genetic reconstruction of dopamine D1 receptor signaling in the nucleus accumbens facilitates natural and drug reward responses.

The dopamine D1 receptor (D1R) facilitates reward acquisition and its alteration leads to profound learning deficits. However, its minimal functional circuit requirement is unknown. Using conditional reconstruction of functional D1R signaling in D1R knock-out mice, we define distinct requirements of D1R in subregions of the nucleus accumbens (NAc) for specific dimensions of reward. We demonstrate that D1R expression in the core region of the NAc (NAc(Core)), but not the shell (NAc(Shell)), enhances selectively a unique form of pavlovian conditioned approach and mediates D1R-dependent cocaine sensitization. However, D1R expression in either the NAc(Core) or the NAc(Shell) improves instrumental responding for reward. In contrast, neither NAc(Core) nor NAc(Shell) D1R is sufficient to promote motivation to work for reward in a progressive ratio task or for motor learning. These results highlight dissociated circuit requirements of D1R for dopamine-dependent behaviors.

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

AAV-mediated interneuron-specific gene replacement for Dravet syndrome.

Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy of SCN1A , which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV human SCN1A gene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form of SCN1A to circumvent AAV packaging limitations and deliver SCN1A via a dual vector approach using cell class-specific enhancers. These constructs produced full-length Na V 1.1 protein and functional sodium channels in HEK293 cells and in brain cells in vivo . After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles of Scn1a, at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves of SCN1A , or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting of SCN1A conferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediated SCN1A gene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

Enhancer viruses for combinatorial cell-subclass-specific labeling.

Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

Roundabout receptor 2 maintains inhibitory control of the adult midbrain.

The maintenance of excitatory and inhibitory balance in the brain is essential for its function. Here we find that the developmental axon guidance receptor Roundabout 2 (Robo2) is critical for the maintenance of inhibitory synapses in the adult ventral tegmental area (VTA), a brain region important for the production of the neurotransmitter dopamine. Following selective genetic inactivation of Robo2 in the adult VTA of mice, reduced inhibitory control results in altered neural activity patterns, enhanced phasic dopamine release, behavioral hyperactivity, associative learning deficits, and a paradoxical inversion of psychostimulant responses. These behavioral phenotypes could be phenocopied by selective inactivation of synaptic transmission from local GABAergic neurons of the VTA, demonstrating an important function for Robo2 in regulating the excitatory and inhibitory balance of the adult brain.

Manipulating gene expression in projection-specific neuronal populations using combinatorial viral approaches.

The mammalian brain contains tremendous structural and genetic complexity that is vital for its function. The elucidation of gene expression profiles in the brain, coupled with the development of large-scale connectivity maps and emerging viral vector-based approaches for target-selective gene manipulation, now allow for detailed dissection of gene-circuit interfaces. This protocol details how to perform combinatorial viral injections to manipulate gene expression in subsets of neurons interconnecting two brain regions. This method utilizes stereotaxic injection of a retrograde transducing CAV2-Cre virus into one brain region, combined with injection of a locally transducing Cre-dependent AAV virus into another brain region. This technique is widely applicable to the genetic dissection of neural circuitry, as it enables selective expression of candidate genes, dominant-negatives, fluorescent reporters, or genetic tools within heterogeneous populations of neurons based upon their projection targets.

Stem cell factor functions as an outgrowth-promoting factor to enable axon exit from the midline intermediate target.

Commissural axons are attracted to the midline intermediate target by chemoattractants, but upon crossing the midline they switch off responsiveness to attractants and switch on responsiveness to midline repellents, which expel the axons from the midline and enable them to move on. Here we show that midline exit also requires the stimulation of axon outgrowth by Stem Cell Factor (SCF, also known as Steel Factor). SCF is expressed by midline floor plate cells, and its receptor Kit, a receptor tyrosine kinase, is expressed by commissural axons. In Steel and Kit mutant mice, the majority of commissural axons line up transiently at the contralateral edge of the floor plate, showing a delay in midline exit. In vitro, SCF selectively promotes outgrowth of postcrossing, but not precrossing, commissural axons. Our findings identify SCF as a guidance cue in the CNS, and provide evidence that exiting intermediate targets requires activation of outgrowth-promoting mechanisms.

Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain.

The division of the mammalian forebrain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic proteins (BMPs) have been implicated in dorsal midline development, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline development in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signaling plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression.

Alternative splicing of the Robo3 axon guidance receptor governs the midline switch from attraction to repulsion.

Alternative splicing provides a means to increase the complexity of gene function in numerous biological processes, including nervous system wiring. Navigating axons switch responses from attraction to repulsion at intermediate targets, allowing them to grow to each intermediate target and then to move on. The mechanisms underlying this switch remain poorly characterized. We previously showed that the Slit receptor Robo3 is required for spinal commissural axons to enter and cross the midline intermediate target. We report here the existence of two functionally antagonistic isoforms of Robo3 with distinct carboxy termini arising from alternative splicing. Robo3.1 is deployed on the precrossing and crossing portions of commissural axons and allows midline crossing by silencing Slit repulsion. Robo3.2 becomes expressed on the postcrossing portion and blocks midline recrossing, favoring Slit repulsion. The tight spatial regulation of opponent splice variants helps ensure high-fidelity transition of axonal responses from attraction to repulsion at the midline.

Secreted sulfatases Sulf1 and Sulf2 have overlapping yet essential roles in mouse neonatal survival.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) use highly sulfated polysaccharide side-chains to interact with several key growth factors and morphogens, thereby regulating their accessibility and biological activity. Various sulfotransferases and sulfatases with differing specificities control the pattern of HSPG sulfation, which is functionally critical. Among these enzymes in the mouse are two secreted 6-O-endosulfatases, Sulf1 and Sulf2, which modify HSPGs in the extracellular matrix and on the cell surface. The roles of Sulf1 and Sulf2 during normal development are not well understood. METHODS/RESULTS: To investigate the importance of Sulf1 and Sulf2 for embryonic development, we generated mice genetically deficient in these genes and assessed the phenotypes of the resulting secreted sulfatase-deficient mice. Surprisingly, despite the established crucial role of HSPG interactions during development, neither Sulf1- nor Sulf2-deficient mice showed significant developmental flaws. In contrast, mice deficient in both Sulf1and Sulf2 exhibited highly penetrant neonatal lethality. Loss of viability was associated with multiple, although subtle, developmental defects, including skeletal and renal abnormalities. CONCLUSIONS: These results show that Sulf1 and Sulf2 play overlapping yet critical roles in mouse development and are redundant and essential for neonatal survival.