Development, wiring and function of dopamine neuron subtypes.

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviours and are linked to various brain diseases. Considerable progress has been made in identifying mDA neuron subtypes, and recent work has begun to unveil how these neuronal subtypes develop and organize into functional brain structures. This progress is important for further understanding the disparate physiological functions of mDA neurons and their selective vulnerability in disease, and will ultimately accelerate therapy development. This Review discusses recent advances in our understanding of molecularly defined mDA neuron subtypes and their circuits, ranging from early developmental events, such as neuron migration and axon guidance, to their wiring and function, and future implications for therapeutic strategies.

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.

An Image-Based miRNA Screen Identifies miRNA-135s As Regulators of CNS Axon Growth and Regeneration by Targeting Krüppel-like Factor 4.

During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migration in vitro and in vivo in male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135-KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration.SIGNIFICANCE STATEMENT Axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135-KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.

DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells.

The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems.

Lrig2 Negatively Regulates Ectodomain Shedding of Axon Guidance Receptors by ADAM Proteases.

Many guidance receptors are proteolytically cleaved by membrane-associated metalloproteases of the ADAM family, leading to the shedding of their ectodomains. Ectodomain shedding is crucial for receptor signaling and function, but how this process is controlled in neurons remains poorly understood. Here, we show that the transmembrane protein Lrig2 negatively regulates ADAM-mediated guidance receptor proteolysis in neurons. Lrig2 binds Neogenin, a receptor for repulsive guidance molecules (RGMs), and prevents premature Neogenin shedding by ADAM17 (TACE). RGMa reduces Lrig2-Neogenin interactions, providing ADAM17 access to Neogenin and allowing this protease to induce ectodomain shedding. Regulation of ADAM17-mediated Neogenin cleavage by Lrig2 is required for neurite growth inhibition by RGMa in vitro and for cortical neuron migration in vivo. Furthermore, knockdown of Lrig2 significantly improves CNS axon regeneration. Together, our data identify a unique ligand-gated mechanism to control receptor shedding by ADAMs and reveal functions for Lrigs in neuron migration and regenerative failure.

TRIM46 Controls Neuronal Polarity and Axon Specification by Driving the Formation of Parallel Microtubule Arrays.

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

Axon guidance proteins in neurological disorders.

Many neurological disorders are characterised by structural changes in neuronal connections, ranging from presymptomatic synaptic changes to the loss or rewiring of entire axon bundles. The molecular mechanisms that underlie this perturbed connectivity are poorly understood, but recent studies suggest a role for axon guidance proteins. Axon guidance proteins guide growing axons during development and control structural plasticity of synaptic connections in adults. Changes in expression or function of these proteins might induce pathological changes in neural circuits that predispose to, or cause, neurological diseases. For some neurological disorders, such as midline crossing disorders, investigators have identified causative mutations in genes for axon guidance. However, for most other disorders, evidence is correlative and further studies are needed to confirm the pathological role of defects in proteins for axon guidance. Importantly, further insight into how dysregulation of axon guidance proteins causes disease will help the development of therapeutic strategies for neurological disorders.

The intracellular redox protein MICAL-1 regulates the development of hippocampal mossy fibre connections.

Mical is a reduction-oxidation (redox) enzyme that functions as an unusual F-actin disassembly factor during Drosophila development. Although three Molecule interacting with CasL (MICAL) proteins exist in vertebrate species, their mechanism of action remains poorly defined and their role in vivo unknown. Here, we report that vertebrate MICAL-1 regulates the targeting of secretory vesicles containing immunoglobulin superfamily cell adhesion molecules (IgCAMs) to the neuronal growth cone membrane through its ability to control the actin cytoskeleton using redox chemistry, thereby maintaining appropriate IgCAM cell surface levels. This precise regulation of IgCAMs by MICAL-1 is essential for the lamina-specific targeting of mossy fibre axons onto CA3 pyramidal neurons in the developing mouse hippocampus in vivo. These findings reveal the first in vivo role for a vertebrate MICAL protein, expand the repertoire of cellular functions controlled through MICAL-mediated effects on the cytoskeleton, and provide insights into the poorly characterized mechanisms underlying neuronal protein cell surface expression and lamina-specific axonal targeting.

Microtubule minus-end binding protein CAMSAP2 controls axon specification and dendrite development.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

Developmental and activity-dependent miRNA expression profiling in primary hippocampal neuron cultures.

MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of ∼22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2-4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes.