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Microglia (μG), the resident immune cells in the central nervous system, surveil the parenchyma to maintain the structural and functional homeostasis of neurons. Besides, they influence neurogenesis and synaptogenesis through complement-mediated phagocytosis. Emerging evidence suggests that μG may also influence development through proinflammatory cytokines. Here, we examined the premise that tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), the two most prominent components of the μG secretome, influence retinal development, specifically the morphological and functional differentiation of human retinal ganglion cells (hRGCs). Using controlled generation of hRGCs and human μG (hμG) from pluripotent stem cells, we demonstrate that TNF-α and IL-1β secreted by unchallenged hμG did not influence hRGC generation. However, their presence significantly facilitated neuritogenesis along with the basal function of hRGCs, which involved the recruitment of the AKT/mTOR pathway. We present ex vivo evidence that proinflammatory cytokines may play an important role in the morphological and physiological maturation of hRGCs, which may be recapitulated for regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca 2+ , across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca 2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca 2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca 2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

In this issue of Neuron, Yamada et al. 1 show that fast excitatory neurotransmission by protons acting at acid-sensing ion channels (ASICs) mediates mechanical force-evoked signaling at the Merkel cell-neurite complex, contributing to mammalian tactile discrimination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Functional cochlear hair cells (HCs) innervated by spiral ganglion neurons (SGNs) are essential for hearing, whereas robust models that recapitulate the peripheral auditory circuity are still lacking. Here, we developed cochlear organoids with functional peripheral auditory circuity in a staging three-dimensional (3D) co-culture system by initially reprogramming cochlear progenitor cells (CPCs) with increased proliferative potency that could be long-term expanded, then stepwise inducing the differentiation of cochlear HCs, as well as the outgrowth of neurites from SGNs. The function of HCs and synapses within organoids was confirmed by a series of morphological and electrophysiological evaluations. Single-cell mRNA sequencing revealed the differentiation trajectories of CPCs toward the major cochlear cell types and the dynamic gene expression during organoid HC development, which resembled the pattern of native HCs. We established the cochlear organoids with functional synapses for the first time, which provides a platform for deciphering the mechanisms of sensorineural hearing loss., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

This protocol provides an improved pipeline for dissociating intact projection neurons from adult mouse cortex for applications including droplet and plate-based single-cell RNA sequencing, qPCR, immunocytochemistry, and long-term in vitro cell culture. This protocol provides a robust and reproducible dissociation pipeline that uses exclusively off-the-shelf reagents, not requiring the use of expensive dissociation kits. The unique incubation steps, in combination with the FACS gating strategy, results in unparalleled enrichment for intact cortical neurons from the adult brain. For complete details on the use and execution of this protocol, please refer to Golan et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K + current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Retinal ganglion cell (RGC) replacement holds potential for restoring vision lost to optic neuropathy. Transplanted RGCs must undergo neuroretinal integration to receive afferent visual signals for processing and efferent transmission. To date, retinal integration following RGC transplantation has been limited. We sought to overcome key barriers to transplanted human stem cell-derived RGC integration. Following co-culture ex vivo on organotypic mouse retinal explants, human RGCs cluster and extend bundled neurites that remain superficial to the neuroretina, hindering afferent synaptogenesis. To enhance integration, we increased the cellular permeability of the internal limiting membrane (ILM). Extracellular matrix digestion using proteolytic enzymes achieved ILM disruption while minimizing retinal toxicity and preserving glial reactivity. ILM disruption is associated with dispersion rather than clustering of co-cultured RGC bodies and neurites, and increased parenchymal neurite ingrowth. The ILM represents a significant obstacle to transplanted RGC connectivity and its circumvention may be necessary for functional RGC replacement., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

UTX, a H3K27me3 demethylase, plays an important role in mouse brain development. However, so little is known about the function of UTX in human neural differentiation and dendritic morphology. In this study, we generated UTX-null human embryonic stem cells using CRISPR/Cas9, and differentiated them into neural progenitor cells and neurons to investigate the effects of UTX loss of function on human neural development. The results showed that the number of differentiated neurons significantly reduced after loss of UTX, and that the dendritic morphology of UTX KO neurons tended to be simplified. The electrophysiological recordings showed that most of the UTX KO neurons were immature. Finally, RNA sequencing identified dozens of differentially expressed genes involved in neural differentiation and synaptic function in UTX KO neurons and our results demonstrated that UTX regulated these critical genes by resolving bivalent promoters. In summary, we establish a reference for the important role of UTX in human neural differentiation and dendritic morphology., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

More than one-half of the interneurons in a mouse olfactory bulb (OB) develop during the first week after birth and predominantly connect to excitatory tufted cells near the superficial granule cell layer (sGCL), unlike late-born interneurons. However, the molecular mechanisms underlying the temporal specification are yet to be identified. In this study, we determined the role of Abelson tyrosine-protein kinase 1 (Abl1) in the temporal development of early-born OB interneurons. Lentiviral knockdown of Abl1 disrupts the sGCL circuit of early-born interneurons through defects in function and circuit integration, resulting in olfactory hyper-sensitivity. We show that doublecortin (Dcx) is phosphorylated by Abl1, which contributes to the stabilization of Dcx, thereby regulating microtubule dynamics. Finally, Dcx overexpression rescues Abl1 knockdown-induced anatomic or functional defects. In summary, specific signaling by Abl1-Dcx in early-born interneurons facilitates the temporal development of the sGCL circuit to regulate innate olfactory functions, such as detection and sensitivity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Voltage-gated calcium channels are exquisitely Ca 2+ selective, conferred primarily by four conserved pore-loop glutamate residues contributing to the selectivity filter. There has been little previous work directly measuring whether the trafficking of calcium channels requires their ability to bind Ca 2+ in the selectivity filter or to conduct Ca 2+ . Here, we examine trafficking of neuronal Ca V 2.1 and 2.2 channels with mutations in their selectivity filter and find reduced trafficking to the cell surface in cell lines. Furthermore, in hippocampal neurons, there is reduced trafficking to the somatic plasma membrane, into neurites, and to presynaptic terminals. However, the Ca V 2.2 selectivity filter mutants are still influenced by auxiliary α 2 δ subunits and, albeit to a reduced extent, by β subunits, indicating the channels are not grossly misfolded. Our results indicate that Ca 2+ binding in the pore of Ca V 2 channels may promote their correct trafficking, in combination with auxiliary subunits. Furthermore, physiological studies utilizing selectivity filter mutant Ca V channels should be interpreted with caution., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3β is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3β. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3β in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV + ) and somatostatin (SST + ) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST + cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development., (Copyright © 2019 UCSF. Published by Elsevier Inc. All rights reserved.)

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca 2+ influx sarco/ER Ca 2+ -ATPase inhibitor thapsigargin (THP). Baseline ER Ca 2+ levels measured with the ER Ca 2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca 2+ influx and decreased intracellular Ca 2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca 2+ entry regulators, with no alterations in ER Ca 2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuromodulators regulate circuits throughout the nervous system, and revealing the cell types and stimulus conditions controlling their release is vital to understanding their function. The effects of the neuromodulator nitric oxide (NO) have been studied in many circuits, including in the vertebrate retina, where it regulates synaptic release, gap junction coupling, and blood vessel dilation, but little is known about the cells that release NO. We show that a single type of amacrine cell (AC) controls NO release in the inner retina, and we report its light responses, electrical properties, and calcium dynamics. We discover that this AC forms a dense gap junction network and that the strength of electrical coupling in the network is regulated by light through NO. A model of the network offers insights into the biophysical specializations leading to auto-regulation of NO release within the network., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Investigations over half a century have indicated that mechanical forces induce neurite growth, with neurites elongating at a rate of 0.1-0.3 μm h -1 pN -1 when mechanical force exceeds a threshold, with this being identified as 400-1000 pN for neurites of PC12 cells. In this article, we demonstrate that neurite elongation of PC12 cells proceeds at the same previously identified rate on application of mechanical tension of ∼1 pN, which is significantly lower than the force generated in vivo by axons and growth cones. This observation raises the possibility that mechanical tension may act as an endogenous signal used by neurons for promoting neurite elongation., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Voltage-gated calcium channel auxiliary α2δ subunits are important for channel trafficking and function. Here, we compare the effects of α2δ-1 and an α2δ-like protein called Cachd1 on neuronal N-type (Ca V 2.2) channels, which are important in neurotransmission. Previous structural studies show the α2δ-1 VWA domain interacting with the first loop in Ca V 1.1 domain-I via its metal ion-dependent adhesion site (MIDAS) motif and additional Cache domain interactions. Cachd1 has a disrupted MIDAS motif. However, Cachd1 increases Ca V 2.2 currents substantially (although less than α2δ-1) and increases Ca V 2.2 cell surface expression by reducing endocytosis. Although the effects of α2δ-1 are abolished by mutation of Asp122 in Ca V 2.2 domain-I, which mediates interaction with its VWA domain, the Cachd1 responses are unaffected. Furthermore, Cachd1 co-immunoprecipitates with Ca V 2.2 and inhibits co-immunoprecipitation of α2δ-1 by Ca V 2.2. Cachd1 also competes with α2δ-1 for effects on trafficking. Thus, Cachd1 influences both Ca V 2.2 trafficking and function and can inhibit responses to α2δ-1., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In mammals, neurons in the peripheral nervous system (PNS) have regenerative capacity following injury, but it is generally absent in the CNS. This difference is attributed, at least in part, to the intrinsic ability of PNS neurons to activate a unique regenerative transcriptional program following injury. Here, we profiled gene expression following sciatic nerve crush in mice and identified long noncoding RNAs (lncRNAs) that act in the regenerating neurons and which are typically not expressed in other contexts. We show that two of these lncRNAs regulate the extent of neuronal outgrowth. We then focus on one of these, Silc1, and show that it regulates neuroregeneration in cultured cells and in vivo, through cis-acting activation of the transcription factor Sox11., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is a neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene. Induced pluripotent stem cell (iPSC) models of HD provide an opportunity to study the mechanisms underlying disease pathology in disease-relevant patient tissues. Murine studies have demonstrated that HTT is intricately involved in corticogenesis. However, the effect of mutant Hungtintin (mtHTT) in human corticogenesis has not yet been thoroughly explored. This examination is critical, due to inherent differences in cortical development and timing between humans and mice. We therefore differentiated HD and non-diseased iPSCs into functional cortical neurons. While HD patient iPSCs can successfully differentiate toward a cortical fate in culture, the resulting neurons display altered transcriptomics, morphological and functional phenotypes indicative of altered corticogenesis in HD., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The brain of Down syndrome (DS) patients exhibits fewer interneurons in the cerebral cortex, but its underlying mechanism remains unknown. By morphometric analysis of cortical interneurons generated from DS and euploid induced pluripotent stem cells (iPSCs), we found that DS GABA neurons are smaller and with fewer neuronal processes. The proportion of calretinin over calbindin GABA neurons is reduced, and the neuronal migration capacity is decreased. Such phenotypes were replicated following transplantation of the DS GABAergic progenitors into the mouse medial septum. Gene expression profiling revealed altered cell migratory pathways, and correction of the PAK1 pathway mitigated the cell migration deficit in vitro. These results suggest that impaired migration of DS GABAergic neurons may contribute to the reduced number of interneurons in the cerebral cortex and hippocampus in DS patients., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Swiprosin-1/Efhd2 (Efhd2) is highly expressed in the CNS during development and in the adult. EFHD2 is regulated by Ca 2+ binding, stabilizes F-actin, and promotes neurite extension. Previous studies indicated a dysregulation of EFHD2 in human Alzheimer's disease brains. We hypothesized a detrimental effect of genetic ablation of Efhd2 on hippocampal integrity and specifically investigated adult hippocampal neurogenesis. Efhd2 was expressed throughout adult neuronal development and in mature neurons. We observed a severe reduction of the survival of adult newborn neurons in Efhd2 knockouts, starting at the early neuroblast stage. Spine formation and dendrite growth of newborn neurons were compromised in full Efhd2 knockouts, but not upon cell-autonomous Efhd2 deletion. Together with our finding of severe hippocampal tauopathy in Efhd2 knockout mice, these data connect Efhd2 to impaired synaptic plasticity as present in Alzheimer's disease and identify a role of Efhd2 in neuronal survival and synaptic integration in the adult hippocampus., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

While the cell-intrinsic pathways governing beige adipocyte development and phenotype have been increasingly delineated, comparatively little is known about how beige adipocytes interact with other cell types in fat. Here, we introduce a whole-tissue clearing method for adipose that permits immunolabeling and three-dimensional profiling of structures including thermogenic adipocytes and sympathetic innervation. We found that tissue architecture and sympathetic innervation differ significantly between subcutaneous and visceral depots. Subcutaneous fat demonstrates prominent regional variation in beige fat biogenesis with localization of UCP1 + beige adipocytes to areas with dense sympathetic neurites. We present evidence that the density of sympathetic projections is dependent on PRDM16 in adipocytes, providing another potential mechanism underlying the metabolic benefits mediated by PRDM16. This powerful imaging tool highlights the interaction of tissue components during beige fat biogenesis and reveals a previously undescribed mode of regulation of the sympathetic nervous system by adipocytes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Our ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei-specific upregulation of Ascl1 after injury. Ascl1 overexpression in DRG neurons of C57BL/6 mice enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is downregulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single-cell RNA- seq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of neurons and vessels shares striking anatomical and molecular features, and it is presumably orchestrated by an overlapping repertoire of extracellular signals. CNS macrophages have been implicated in various developmental functions, including the morphogenesis of neurons and vessels. However, whether CNS macrophages can coordinately influence neurovascular development and the identity of the signals involved therein is unclear. Here, we demonstrate that activity of the cell surface receptor CD95 regulates neuronal and vascular morphogenesis in the post-natal brain and retina. Furthermore, we identify CNS macrophages as the main source of CD95L, and macrophage-specific deletion thereof reduces both neurovascular complexity and synaptic activity in the brain. CD95L-induced neuronal and vascular growth is mediated through src-family kinase (SFK) and PI3K signaling. Together, our study highlights a coordinated neurovascular development instructed by CNS macrophage-derived CD95L, and it underlines the importance of macrophages for the establishment of the neurovascular network during CNS development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Functional deficits persist after spinal cord injury (SCI) because axons in the adult mammalian central nervous system (CNS) fail to regenerate. However, modest levels of spontaneous functional recovery are typically observed after trauma and are thought to be mediated by the plasticity of intact circuitry. The mechanisms underlying intact circuit plasticity are not delineated. Here, we characterize the in vivo transcriptome of sprouting intact neurons from Ngr1 null mice after partial SCI. We identify the lysophosphatidic acid signaling modulators LPPR1 and LPAR1 as intrinsic axon growth modulators for intact corticospinal motor neurons after adjacent injury. Furthermore, in vivo LPAR1 inhibition or LPPR1 overexpression enhances sprouting of intact corticospinal tract axons and yields greater functional recovery after unilateral brainstem lesion in wild-type mice. Thus, the transcriptional profile of injury-induced sprouting of intact neurons reveals targets for therapeutic enhancement of axon growth initiation and new synapse formation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The maturation of intercellular adhesion is an essential process for establishing the signal transduction network in living cells. Although the maturation is naturally considered to enhance the signal transduction, the relationship between the signal transduction and the maturation process has not been revealed in detail using time-course data. Here, using a coculture of mast cells and neurites, differences in maturation between individual cells were estimated as a function of the adhesion strength by our original single-cell measurement method utilizing a laser-induced impulsive force. When an intense femtosecond laser is focused into a culture medium under a microscope, shock and stress waves are generated at the laser focal point that exert an impulsive force on individual cells. In our method, this impulse is used to break the adhesion between a mast cell and a neurite. The magnitude of the impulse is then quantified by a local force-measurement system utilizing an atomic force microscope, and the adhesion strength is estimated from the threshold of the impulse required to break the adhesion. The measurement is conducted within 1 min/cell, and thus, data on the individual differences of the adhesion strength can be obtained within only a few hours. Coculturing of neurites and mast cells for 4 h resulted in a specific adhesion that was stronger than the nonspecific adhesions between the substrate and mast cells. In the time-course investigation, we identified two distinct temporal patterns of adhesion: 1) the strength at 24 h was the same as the initial strength; and 2) the strength increased threefold from baseline and became saturated within 24 h. Based on these results, the distribution of CADM1 adhesion molecules in the neurites was suggested to be inhomogeneous, and the relationship between adhesion maturation and the signal-transduction process was considered., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis, we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro, we generated neurons harboring disease-causing mutations from patient-specific, induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons, reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro, including oxidative stress vulnerability and altered neuronal activity. Notably, PD neurons show that elevated RBFOX1, a gene previously linked to neurodevelopmental diseases, underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytoplasmic dynein, the major motor driving retrograde axonal transport, must be actively localized to axon terminals. This localization is critical as dynein powers essential retrograde trafficking events required for neuronal survival, such as neurotrophic signaling. Here, we demonstrate that the outward transport of dynein from soma to axon terminal is driven by direct interactions with the anterograde motor kinesin-1. In developing neurons, we find that dynein dynamically cycles between neurites, following kinesin-1 and accumulating in the nascent axon coincident with axon specification. In established axons, dynein is constantly transported down the axon at slow axonal transport speeds; inhibition of the kinesin-1-dynein interaction effectively blocks this process. In vitro and live-imaging assays to investigate the underlying mechanism lead us to propose a new model for the slow axonal transport of cytosolic cargos, based on short-lived direct interactions of cargo with a highly processive anterograde motor. VIDEO ABSTRACT., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Proneural proteins of the class I/II basic-helix-loop-helix (bHLH) family are highly conserved transcription factors. Class I bHLH proteins are expressed in a broad number of tissues during development, whereas class II bHLH protein expression is more tissue restricted. Our understanding of the function of class I/II bHLH transcription factors in both invertebrate and vertebrate neurobiology is largely focused on their function as regulators of neurogenesis. Here, we show that the class I bHLH proteins Daughterless and Tcf4 are expressed in postmitotic neurons in Drosophila melanogaster and mice, respectively, where they function to restrict neurite branching and synapse formation. Our data indicate that Daughterless performs this function in part by restricting the expression of the cell adhesion molecule Neurexin. This suggests a role for these proteins outside of their established roles in neurogenesis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most-common genetic determinants of Parkinson's disease (PD). The G2019S mutation is detected most frequently and is associated with increased kinase activity. Whereas G2019S mutant dopamine neurons exhibit neurite elongation deficits, the effect of G2019S on other neuronal subtypes is unknown. As PD patients also suffer from non-motor symptoms that may be unrelated to dopamine neuron loss, we used induced pluripotent stem cells (iPSCs) to assess morphological and functional properties of peripheral sensory neurons. LRRK2 G2019S iPSC-derived sensory neurons exhibited normal neurite length but had large microtubule-containing neurite aggregations. Additionally, LRRK2 G2019S iPSC-derived sensory neurons displayed altered calcium dynamics. Treatment with LRRK2 kinase inhibitors resulted in significant, but not complete, morphological and functional rescue. These data indicate a role for LRRK2 kinase activity in sensory neuron structure and function, which when disrupted, may lead to sensory neuron deficits in PD., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthesis and release of neurotransmitters such as acetylcholine (ACh) are key to synaptic function. However, little is known about the spatial regulation of their synthesizing machinery. Here, we demonstrate that ataxia-related protein BNIP-H/Caytaxin links kinesin-1 (KLC1) to ATP citrate lyase (ACL), a key enzyme for ACh synthesis, and transports it toward neurite terminals. There, BNIP-H/ACL complex synergistically recruits another enzyme choline acetyltransferase (ChAT), leading to enhanced secretion of ACh. ACh then activates MAPK/ERK via muscarinic receptors to promote neurite outgrowth. In mice deficient in BNIP-H, ACL fails to interact with KLC1, and formation of the ACL/ChAT complex is prevented, whereas the disease-associated BNIP-H mutation fails to target ACL for neurite outgrowth. Significantly, Bnip-h knockdown in zebrafish causes developmental defect in motor neurons through impaired cholinergic pathway, leading to motor disorder. Therefore, precise targeting of the cholinergic machinery through BNIP-H is essential for the local production of ACh for morphogenesis and neurotransmission., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A striking neuronal connectivity change in C. elegans involves the coordinated elimination of existing synapses and formation of synapses at new locations, without altering neuronal morphology. Here, we investigate the tripartite interaction between dynamic microtubules (MTs), kinesin-1, and vesicular cargo during this synapse remodeling. We find that a reduction in the dynamic MT population in motor neuron axons, resulting from genetic interaction between loss of function in the conserved MAPKKK dlk-1 and an α-tubulin mutation, specifically blocks synapse remodeling. Using live imaging and pharmacological modulation of the MT cytoskeleton, we show that dynamic MTs are increased at the onset of remodeling and are critical for new synapse formation. DLK-1 acts during synapse remodeling, and its function involves MT catastrophe factors including kinesin-13/KLP-7 and spastin/SPAS-1. Through a forward genetic screen, we identify gain-of-function mutations in kinesin-1 that can compensate for reduced dynamic MTs to promote synaptic vesicle transport during remodeling. Our data provide in vivo evidence supporting the requirement of dynamic MTs for kinesin-1-dependent axonal transport and shed light on the role of the MT cytoskeleton in facilitating neural circuit plasticity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)