Teneurin-3 regulates the generation of non-image-forming visual circuitry and responsiveness to light in the suprachiasmatic nucleus.

Visual system function depends upon the elaboration of precise connections between retinal ganglion cell (RGC) axons and their central targets in the brain. Though some progress has been made in defining the molecules that regulate RGC connectivity required for the assembly and function of image-forming circuitry, surprisingly little is known about factors required for intrinsically photosensitive RGCs (ipRGCs) to target a principal component of the non-image-forming circuitry: the suprachiasmatic nucleus (SCN). Furthermore, the molecules required for forming circuits critical for circadian behaviors within the SCN are not known. We observe here that the adhesion molecule teneurin-3 (Tenm3) is highly expressed in vasoactive intestinal peptide (VIP) neurons located in the core region of the SCN. Since Tenm3 is required for other aspects of mammalian visual system development, we investigate roles for Tenm3 in regulating ipRGC-SCN connectivity and function. Our results show that Tenm3 negatively regulates association between VIP and arginine vasopressin (AVP) neurons within the SCN and is essential for M1 ipRGC axon innervation to the SCN. Specifically, in Tenm3-/- mice, we find a reduction in ventro-medial innervation to the SCN. Despite this reduction, Tenm3-/- mice have higher sensitivity to light and faster re-entrainment to phase advances, probably due to the increased association between VIP and AVP neurons. These data show that Tenm3 plays key roles in elaborating non-image-forming visual system circuitry and that it influences murine responses to phase-advancing light stimuli.

The Retinal Ganglion Cell Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration Consortium.

Purpose: The Retinal Ganglion Cell (RGC) Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) consortium was founded in 2021 to help address the numerous scientific and clinical obstacles that impede development of vision-restorative treatments for patients with optic neuropathies. The goals of the RReSTORe consortium are: (1) to define and prioritize the most critical challenges and questions related to RGC regeneration; (2) to brainstorm innovative tools and experimental approaches to meet these challenges; and (3) to foster opportunities for collaborative scientific research among diverse investigators. Design and Participants: The RReSTORe consortium currently includes > 220 members spanning all career stages worldwide and is directed by an organizing committee comprised of 15 leading scientists and physician-scientists of diverse backgrounds. Methods: Herein, we describe the structure and organization of the RReSTORe consortium, its activities to date, and the perceived impact that the consortium has had on the field based on a survey of participants. Results: In addition to helping propel the field of regenerative medicine as applied to optic neuropathies, the RReSTORe consortium serves as a framework for developing large collaborative groups aimed at tackling audacious goals that may be expanded beyond ophthalmology and vision science. Conclusions: The development of innovative interventions capable of restoring vision for patients suffering from optic neuropathy would be transformative for the ophthalmology field, and may set the stage for functional restoration in other central nervous system disorders. By coordinating large-scale, international collaborations among scientists with diverse and complementary expertise, we are confident that the RReSTORe consortium will help to accelerate the field toward clinical translation. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium.

Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system's limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium's efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases.

Secreted semaphorin 3F (Sema3F) and semaphorin 3A (Sema3A) exhibit remarkably distinct effects on deep layer excitatory cortical pyramidal neurons; Sema3F mediates dendritic spine pruning, whereas Sema3A promotes the elaboration of basal dendrites. Sema3F and Sema3A signal through distinct holoreceptors that include neuropilin-2 (Nrp2)/plexinA3 (PlexA3) and neuropilin-1 (Nrp1)/PlexA4, respectively. We find that Nrp2 and Nrp1 are S-palmitoylated in cortical neurons and that palmitoylation of select Nrp2 cysteines is required for its proper subcellular localization, cell surface clustering, and also for Sema3F/Nrp2-dependent dendritic spine pruning in cortical neurons, both in vitro and in vivo. Moreover, we show that the palmitoyl acyltransferase ZDHHC15 is required for Nrp2 palmitoylation and Sema3F/Nrp2-dependent dendritic spine pruning, but it is dispensable for Nrp1 palmitoylation and Sema3A/Nrp1-dependent basal dendritic elaboration. Therefore, palmitoyl acyltransferase-substrate specificity is essential for establishing compartmentalized neuronal structure and functional responses to extrinsic guidance cues.

Netrin-Mediated Axon Guidance to the CNS Midline Revisited.

Varadarajan et al. (2017)-in this issue of Neuron-and Dominici et al. (2017)-published online at Nature-independently show that floor plate-derived netrin-1 is dispensable for commissural neuron axon guidance to the CNS midline during development.

Cas adaptor proteins organize the retinal ganglion cell layer downstream of integrin signaling.

Stratification of retinal neuronal cell bodies and lamination of their processes provide a scaffold upon which neural circuits can be built. However, the molecular mechanisms that direct retinal ganglion cells (RGCs) to resolve into a single-cell retinal ganglion cell layer (GCL) are not well understood. The extracellular matrix protein laminin conveys spatial information that instructs the migration, process outgrowth, and reorganization of GCL cells. Here, we show that the ß1-Integrin laminin receptor is required for RGC positioning and reorganization into a single-cell GCL layer. ß1-Integrin signaling within migrating GCL cells requires Cas signaling-adaptor proteins, and in the absence of ß1-Integrin or Cas function retinal neurons form ectopic cell clusters beyond the inner-limiting membrane (ILM), phenocopying laminin mutants. These data reveal an essential role for Cas adaptor proteins in ß1-Integrin-mediated signaling events critical for the formation of the single-cell GCL in the mammalian retina.

The Control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in drosophila.

Transmembrane semaphorins (Semas) serve evolutionarily conserved guidance roles, and some function as both ligands and receptors. However, the molecular mechanisms underlying the transduction of these signals to the cytoskeleton remain largely unknown. We have identified two direct regulators of Rho family small GTPases, pebble (a Rho guanine nucleotide exchange factor [GEF]) and RhoGAPp190 (a GTPase activating protein [GAP]), that show robust interactions with the cytoplasmic domain of the Drosophila Sema-1a protein. Neuronal pebble and RhoGAPp190 are required to control motor axon defasciculation at specific pathway choice points and also for target recognition during Drosophila neuromuscular development. Sema-1a-mediated motor axon defasciculation is promoted by pebble and inhibited by RhoGAPp190. Genetic analyses show that opposing pebble and RhoGAPp190 functions mediate Sema-1a reverse signaling through the regulation of Rho1 activity. Therefore, pebble and RhoGAPp190 transduce transmembrane semaphorin-mediated guidance cue information that regulates the establishment of neuronal connectivity during Drosophila development.

The RacGAP ß2-Chimaerin selectively mediates axonal pruning in the hippocampus.

Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) ß2-Chimaerin (ß2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds ß2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. ß2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/ß2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development.

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. Here, we identify a Drosophila Cas (DCas), find that Cas proteins are highly expressed in neurons and show that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events.

Semaphorin 7A initiates T-cell-mediated inflammatory responses through alpha1beta1 integrin.

Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through beta1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through alpha1beta1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and alpha1beta1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.

Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve.

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, we now demonstrate that the behavior of somata and axons of the facial nerve is regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, we now show that it is not required for the pathfinding of their somata. Vice versa, we find that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate, for the first time, that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor.

Nervy links protein kinase a to plexin-mediated semaphorin repulsion.

Cyclic nucleotides regulate axonal responses to a number of guidance cues through unknown molecular events. We report here that Drosophila nervy, a member of the myeloid translocation gene family of A kinase anchoring proteins (AKAPs), regulates repulsive axon guidance by linking the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA). Nervy and PKA antagonize Sema-1a-PlexA-mediated repulsion, and the AKAP binding region of Nervy is critical for this effect. Thus, Nervy couples cAMP-PKA signaling to PlexA to regulate Sema-1a-mediated axonal repulsion, revealing a simple molecular mechanism that allows growing axons to integrate inputs from multiple guidance cues.

Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.

Neuropilin-1 (Npn-1) is a receptor that binds multiple ligands from structurally distinct families, including secreted semaphorins (Sema) and vascular endothelial growth factors (VEGF). We generated npn-1 knockin mice, which express an altered ligand binding site variant of Npn-1, and npn-1 conditional null mice to establish the cell-type- and ligand specificity of Npn-1 function in the developing cardiovascular and nervous systems. Our results show that VEGF-Npn-1 signaling in endothelial cells is required for angiogenesis. In striking contrast, Sema-Npn-1 signaling is not essential for general vascular development but is required for axonal pathfinding by several populations of neurons in the CNS and PNS. Remarkably, both Sema-Npn-1 signaling and VEGF-Npn-1 signaling are critical for heart development. Therefore, Npn-1 is a multifunctional receptor that mediates the activities of structurally distinct ligands during development of the heart, vasculature, and nervous system.

Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165.

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.