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.

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.

Best practice standards for circular RNA research.

Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience. This Perspective will specifically address the major class of circRNAs in Eukarya that are generated by a spliceosome-catalyzed back-splicing event. We hope that the implementation of best practice principles for circRNA research will help move the field forward and allow a better functional understanding of this fascinating group of RNAs.

The mouse brain after foot shock in four dimensions: Temporal dynamics at a single-cell resolution.

Acute stress leads to sequential activation of functional brain networks. A biologically relevant question is exactly which (single) cells belonging to brain networks are changed in activity over time after acute stress across the entire brain. We developed a preprocessing and analytical pipeline to chart whole-brain immediate early genes' expression-as proxy for cellular activity-after a single stressful foot shock in four dimensions: that is, from functional networks up to three-dimensional (3D) single-cell resolution and over time. The pipeline is available as an R package. Most brain areas (96%) showed increased numbers of c-fos+ cells after foot shock, yet hypothalamic areas stood out as being most active and prompt in their activation, followed by amygdalar, prefrontal, hippocampal, and finally, thalamic areas. At the cellular level, c-fos+ density clearly shifted over time across subareas, as illustrated for the basolateral amygdala. Moreover, some brain areas showed increased numbers of c-fos+ cells, while others-like the dentate gyrus-dramatically increased c-fos intensity in just a subset of cells, reminiscent of engrams; importantly, this "strategy" changed after foot shock in half of the brain areas. One of the strengths of our approach is that single-cell data were simultaneously examined across all of the 90 brain areas and can be visualized in 3D in our interactive web portal.

Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

Structural basis of semaphorin-plexin cis interaction.

Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns.

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli.

Prebiotic diet normalizes aberrant immune and behavioral phenotypes in a mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by deficits in communication and behavior. Increasing evidence suggests that the microbiota-gut-brain axis and the likely related immune imbalance may play a role in the development of this disorder. Gastrointestinal deficits and gut microbiota dysfunction have been linked to the development or severity of autistic behavior. Therefore, treatments that focus on specific diets may improve gastrointestinal function and aberrant behavior in individuals with ASD. In this study, we investigated whether a diet containing specific prebiotic fibers, namely, 3% galacto-oligosaccharide/fructo-oligosaccharide (GOS/FOS; 9:1), can mitigate the adverse effects of in utero exposure to valproic acid (VPA) in mice. Pregnant BALB/cByJ dams were injected with VPA (600 mg/kg, sc.) or phosphate-buffered saline (PBS) on gestational day 11 (G11). Male offspring were divided into four groups: (1) in utero PBS-exposed with a control diet, (2) in utero PBS-exposed with GOS/FOS diet, (3) in utero VPA-exposed with a control diet, and (4) in utero VPA-exposed with GOS/FOS diet. Dietary intervention started from birth and continued throughout the duration of the experiment. We showed that the prebiotic diet normalized VPA-induced alterations in male offspring, including restoration of key microbial taxa, intestinal permeability, peripheral immune homeostasis, reduction of neuroinflammation in the cerebellum, and impairments in social behavior and cognition in mice. Overall, our research provides valuable insights into the gut-brain axis involvement in ASD development. In addition, dietary interventions might correct the disbalance in gut microbiota and immune responses and, ultimately, might improve detrimental behavioral outcomes in ASD.

UNC13A in amyotrophic lateral sclerosis: from genetic association to therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options and an incompletely understood pathophysiology. Although genomewide association studies (GWAS) have advanced our understanding of the disease, the precise manner in which risk polymorphisms contribute to disease pathogenesis remains unclear. Of relevance, GWAS have shown that a polymorphism (rs12608932) in the UNC13A gene is associated with risk for both ALS and frontotemporal dementia (FTD). Homozygosity for the C-allele at rs12608932 modifies the ALS phenotype, as these patients are more likely to have bulbar-onset disease, cognitive impairment and FTD at baseline as well as shorter survival. UNC13A is expressed in neuronal tissue and is involved in maintaining synaptic active zones, by enabling the priming and docking of synaptic vesicles. In the absence of functional TDP-43, risk variants in UNC13A lead to the inclusion of a cryptic exon in UNC13A messenger RNA, subsequently leading to nonsense mediated decay, with loss of functional protein. Depletion of UNC13A leads to impaired neurotransmission. Recent discoveries have identified UNC13A as a potential target for therapy development in ALS, with a confirmatory trial with lithium carbonate in UNC13A cases now underway and future approaches with antisense oligonucleotides currently under consideration. Considering UNC13A is a potent phenotypic modifier, it may also impact clinical trial outcomes. This present review describes the path from the initial discovery of UNC13A as a risk gene in ALS to the current therapeutic options being explored and how knowledge of its distinct phenotype needs to be taken into account in future trials.

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy.

Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapies.

Long non-coding RNAs in motor neuron development and disease.

Long non-coding RNAs (lncRNAs) are RNAs that exceed 200 nucleotides in length and that are not translated into proteins. Thousands of lncRNAs have been identified with functions in processes such as transcription and translation regulation, RNA processing, and RNA and protein sponging. LncRNAs show prominent expression in the nervous system and have been implicated in neural development, function and disease. Recent work has begun to report on the expression and roles of lncRNAs in motor neurons (MNs). The cell bodies of MNs are located in cortex, brainstem or spinal cord and their axons project into the brainstem, spinal cord or towards peripheral muscles, thereby controlling important functions such as movement, breathing and swallowing. Degeneration of MNs is a pathological hallmark of diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. LncRNAs influence several aspects of MN development and disruptions in these lncRNA-mediated effects are proposed to contribute to the pathogenic mechanisms underlying MN diseases (MNDs). Accumulating evidence suggests that lncRNAs may comprise valuable therapeutic targets for different MNDs. In this review, we discuss the role of lncRNAs (including circular RNAs [circRNAs]) in the development of MNs, discuss how lncRNAs may contribute to MNDs and provide directions for future research.

Neuromuscular junction-on-a-chip: ALS disease modeling and read-out development in microfluidic devices.

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease affecting upper and lower motor neurons with no cure available. Clinical and animal studies reveal that the neuromuscular junction (NMJ), a synaptic connection between motor neurons and skeletal muscle fibers, is highly vulnerable in ALS and suggest that NMJ defects may occur at the early stages of the disease. However, mechanistic insight into how NMJ dysfunction relates to the onset and progression of ALS is incomplete, which hampers therapy development. This is, in part, caused by a lack of robust in vitro models. The ability to combine microfluidic and induced pluripotent stem cell (iPSC) technologies has opened up new avenues for studying molecular and cellular ALS phenotypes in vitro. Microfluidic devices offer several advantages over traditional culture approaches when modeling the NMJ, such as the spatial separation of different cell types and increased control over the cellular microenvironment. Moreover, they are compatible with 3D cell culture, which enhances NMJ functionality and maturity. Here, we review how microfluidic technology is currently being employed to develop more reliable in vitro NMJ models. To validate and phenotype such models, various morphological and functional read-outs have been developed. We describe and discuss the relevance of these read-outs and specifically illustrate how these read-outs have enhanced our understanding of NMJ pathology in ALS. Finally, we share our view on potential future directions and challenges.

Sensory Axon Growth Requires Spatiotemporal Integration of CaSR and TrkB Signaling.

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development.SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system.

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy.

Deciphering the Proteome Dynamics during Development of Neurons Derived from Induced Pluripotent Stem Cells.

Neuronal development is a complex multistep process that shapes neurons by progressing though several typical stages, including axon outgrowth, dendrite formation, and synaptogenesis. Knowledge of the mechanisms of neuronal development is mostly derived from the study of animal models. Advances in stem cell technology now enable us to generate neurons from human induced pluripotent stem cells (iPSCs). Here we provide a mass spectrometry-based quantitative proteomic signature of human iPSC-derived neurons, i.e., iPSC-derived induced glutamatergic neurons and iPSC-derived motor neurons, throughout neuronal differentiation. Tandem mass tag 10-plex labeling was carried out to perform proteomic profiling of cells at different time points. Our analysis reveals significant expression changes (FDR < 0.001) of several key proteins during the differentiation process, e.g., proteins involved in the Wnt and Notch signaling pathways. Overall, our data provide a rich resource of information on protein expression during human iPSC neuron differentiation.

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.

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.