PM2.5 induces developmental neurotoxicity in cortical organoids.

There is mounting evidence implicating the potential neurotoxic effects of PM2.5 during brain development, as it has been observed to traverse both the placental barrier and the fetal blood-brain barrier. However, the current utilization of 2D cell culture and animal models falls short in providing an accurate representation of human brain development. Consequently, the precise mechanisms underlying PM2.5-induced developmental neurotoxicity in humans remain obscure. To address this research gap, we constructed three-dimensional (3D) cortical organoids that faithfully recapitulate the initial stages of human cerebral cortex development. Our goal is to investigate the mechanisms of PM2.5-induced neurotoxicity using 3D brain organoids that express cortical layer proteins. Our findings demonstrate that exposure to PM2.5 concentrations of 5 µg/mL and 50 µg/mL induces neuronal apoptosis and disrupts normal neural differentiation, thereby suggesting a detrimental impact on neurodevelopment. Furthermore, transcriptomic analysis revealed PM2.5 exposure induced aberrations in mitochondrial complex I functionality, which is reminiscent of Parkinson's syndrome, potentially mediated by misguided axon guidance and compromised synaptic maintenance. This study is a pioneering assessment of the neurotoxicity of PM2.5 pollution on human brain tissues based on 3D cortical organoids, and the results are of great significance in guiding the formulation of the next air pollution prevention and control policies in China to achieve the sustainable improvement of air quality and to formulate pollution abatement strategies that can maximize the benefits to public health.

Gradient conducting polymer surfaces with netrin-1-conjugation promote axon guidance and neuron transmission of human iPSC-derived retinal ganglion cells.

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.

A cell-autonomous role for primary cilium-mediated signaling in long-range commissural axon guidance.

Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point or intermediate target. Using different animal models and in vivo, ex vivo and in vitro experiments, we provide evidence for a crucial role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons.

Protocadherin 19 regulates axon guidance in the developing Xenopus retinotectal pathway.

Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons.

An "R-spondin code" for multimodal signaling ON-OFF states.

R-spondins (RSPOs) are a family of secreted proteins and stem cell growth factors that are potent co-activators of Wnt signaling. Recently, RSPO2 and RSPO3 were shown to be multifunctional, not only amplifying Wnt- but also binding BMP- and FGF receptors to downregulate signaling. The common mechanism underlying these diverse functions is that RSPO2 and RSPO3 act as "endocytosers" that link transmembrane proteins to ZNRF3/RNF43 E3 ligases and trigger target internalization. Thus, RSPOs are natural protein targeting chimeras for cell surface proteins. Conducting data mining and cell surface binding assays we report additional candidate RSPO targets, including SMO, PTC1,2, LGI1, ROBO4, and PTPR(F/S). We propose that there is an "R-spondin code" that imparts combinatorial signaling ON-OFF states of multiple growth factors. This code involves the modular RSPO domains, notably distinct motifs in the divergent RSPO-TSP1 domains to mediate target interaction and internalization. The RSPO code offers a novel framework for the understanding how diverse signaling pathways may be coordinately regulated in development and disease.

Gestational VPA exposure reduces the density of juxtapositions between TH+ axons and calretinin or calbindin expressing cells in the ventrobasal forebrain of neonatal mice.

Gestational exposure to valproic acid (VPA) is a valid rodent model of human autism spectrum disorder (ASD). VPA treatment is known to bring about specific behavioral deficits of sociability, matching similar alterations in human autism. Previous quantitative morphometric studies from our laboratory showed a marked reduction and defasciculation of the mesotelencephalic dopaminergic pathway of VPA treated mice, along with a decrease in tissue dopamine in the nucleus accumbens (NAc), but not in the caudatoputamen (CPu). In the present study, the correlative distribution of tyrosine hydroxylase positive (TH+) putative axon terminals, presynaptic to the target neurons containing calretinin (CR) or calbindin (CB), was assessed using double fluorescent immunocytochemistry and confocal laser microscopy in two dopamine recipient forebrain regions, NAc and olfactory tubercle (OT) of neonatal mice (mothers injected with VPA on ED13.5, pups investigated on PD7). Representative image stacks were volumetrically analyzed for spatial proximity and abundance of presynaptic (TH+) and postsynaptic (CR+, CB+) structures with the help of an Imaris (Bitplane) software. In VPA mice, TH/CR juxtapositions were reduced in the NAc, whereas the TH/CB juxtapositions were impoverished in OT. Volume ratios of CR+ and CB+ elements remained unchanged in NAc, whereas that of CB+ was markedly reduced in OT; here the abundance of TH+ axons was also diminished. CR and CB were found to partially colocalize with TH in the VTA and SN. In VPA exposed mice, the abundance of CR+ (but not CB+) perikarya increased both in VTA and SN, however, this upregulation was not mirrored by an increase of the number of CR+/TH+ double labeled cells. The observed reduction of total CB (but not of CB+ perikarya) in the OT of VPA exposed animals signifies a diminished probability of synaptic contacts with afferent TH+ axons, presumably by reducing the available synaptic surface. Altered dopaminergic input to ventrobasal forebrain targets during late embryonic development will likely perturb the development and consolidation of neural and synaptic architecture, resulting in lasting changes of the neuronal patterning (detected here as reduced synaptic input to dopaminoceptive interneurons) in ventrobasal forebrain regions specifically involved in motivation and reward.

Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance.

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.

Rac1 and Nectin3 are essential for PCP-directed axon guidance in the peripheral auditory system.

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through in vivo genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea.

ß-arrestins Are Scaffolding Proteins Required for Shh-Mediated Axon Guidance.

During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that ß-arrestin1 and ß-arrestin2 (ß-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that ß-arrestins are expressed in rat commissural neurons. We also found that Smo, ß-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on ß-arrestins. ß-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that ß-arrestins are required to activate Src kinase downstream of Shh. ß-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative ß-arrestins, ß-arrestin1 V53D which blocks the internalization of Smo and ß-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative ß-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that ß-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.

Placental growth factor promotes neural invasion and predicts disease prognosis in resectable pancreatic cancer.

BACKGROUND: Surgery represents the only curative treatment option for pancreatic ductal adenocarcinoma (PDAC), but recurrence in more than 85% of patients limits the success of curative-intent tumor resection. Neural invasion (NI), particularly the spread of tumor cells along nerves into extratumoral regions of the pancreas, constitutes a well-recognized risk factor for recurrence. Hence, monitoring and therapeutic targeting of NI offer the potential to stratify recurrence risk and improve recurrence-free survival. Based on the evolutionary conserved dual function of axon and vessel guidance molecules, we hypothesize that the proangiogenic vessel guidance factor placental growth factor (PlGF) fosters NI. To test this hypothesis, we correlated PlGF with NI in PDAC patient samples and functionally assessed its role for the interaction of tumor cells with nerves. METHODS: Serum levels of PlGF and its soluble receptor sFlt1, and expression of PlGF mRNA transcripts in tumor tissues were determined by ELISA or qPCR in a retrospective discovery and a prospective validation cohort. Free circulating PlGF was calculated from the ratio PlGF/sFlt1. Incidence and extent of NI were quantified based on histomorphometric measurements and separately assessed for intratumoral and extratumoral nerves. PlGF function on reciprocal chemoattraction and directed neurite outgrowth was evaluated in co-cultures of PDAC cells with primary dorsal-root-ganglia neurons or Schwann cells using blocking anti-PlGF antibodies. RESULTS: Elevated circulating levels of free PlGF correlated with NI and shorter overall survival in patients with PDAC qualifying for curative-intent surgery. Furthermore, high tissue PlGF mRNA transcript levels in patients undergoing curative-intent surgery correlated with a higher incidence and greater extent of NI spreading to tumor-distant extratumoral nerves. In turn, more abundant extratumoral NI predicted shorter disease-free and overall survival. Experimentally, PlGF facilitated directional and dynamic changes in neurite outgrowth of primary dorsal-root-ganglia neurons upon exposure to PDAC derived guidance and growth factors and supported mutual chemoattraction of tumor cells with neurons and Schwann cells. CONCLUSION: Our translational results highlight PlGF as an axon guidance factor, which fosters neurite outgrowth and attracts tumor cells towards nerves. Hence, PlGF represents a promising circulating biomarker of NI and potential therapeutic target to improve the clinical outcome for patients with resectable PDAC.

Insertion of a neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development.

During development of the nervous system, neurons connect to one another in a precisely organized manner. Sensory systems provide a good example of this organization, whereby the composition of the outside world is represented in the brain by neuronal maps. Establishing correct patterns of neural circuitry is crucial, as inaccurate map formation can lead to severe disruptions in sensory processing. In rodents, olfactory stimuli modulate a wide variety of behaviors essential for survival. The formation of the olfactory glomerular map is dependent on molecular cues that guide olfactory receptor neuron axons to broad regions of the olfactory bulb and on cell adhesion molecules that promote axonal sorting into specific synaptic units in this structure. Here, we demonstrate that the cell adhesion molecule Amigo1 is expressed in a subpopulation of olfactory receptor neurons, and we investigate its role in the precise targeting of olfactory receptor neuron axons to the olfactory bulb using a genetic loss-of-function approach in mice. While ablation of Amigo1 did not lead to alterations in olfactory sensory neuron axonal targeting, our experiments revealed that the presence of a neomycin resistance selection cassette in the Amigo1 locus can lead to off-target effects that are not due to loss of Amigo1 expression, including unexpected altered gene expression in olfactory receptor neurons and reduced glomerular size in the ventral region of the olfactory bulb. Our results demonstrate that insertion of a neomycin selection cassette into the mouse genome can have specific deleterious effects on the development of the olfactory system and highlight the importance of removing antibiotic resistance cassettes from genetic loss-of-function mouse models when studying olfactory system development.

Activity-dependent formation of the topographic map and the critical period in the development of mammalian olfactory system.

Neural activity influences every aspect of nervous system development. In olfactory systems, sensory neurons expressing the same odorant receptor project their axons to stereotypically positioned glomeruli, forming a spatial map of odorant receptors in the olfactory bulb. As individual odors activate unique combinations of glomeruli, this map forms the basis for encoding olfactory information. The establishment of this stereotypical olfactory map requires coordinated regulation of axon guidance molecules instructed by spontaneous activity. Recent studies show that sensory experiences also modify innervation patterns in the olfactory bulb, especially during a critical period of the olfactory system development. This review examines evidence in the field to suggest potential mechanisms by which various aspects of neural activity regulate axon targeting. We also discuss the precise functions served by neural plasticity during the critical period.

Plexin B3 guides axons to cross the midline in vivo.

During the development of neural circuits, axons are guided by a variety of molecular cues to navigate through the brain and establish precise connections with correct partners at the right time and place. Many axon guidance cues have been identified and they play pleiotropic roles in not only axon guidance but also axon fasciculation, axon pruning, and synaptogenesis as well as cell migration, angiogenesis, and bone formation. In search of receptors for Sema3E in axon guidance, we unexpectedly found that Plexin B3 is highly expressed in retinal ganglion cells of zebrafish embryos when retinal axons are crossing the midline to form the chiasm. Plexin B3 has been characterized to be related to neurodevelopmental disorders. However, the investigation of its pathological mechanisms is hampered by the lack of appropriate animal model. We provide evidence that Plexin B3 is critical for axon guidance in vivo. Plexin B3 might function as a receptor for Sema3E while Neuropilin1 could be a co-receptor. The intracellular domain of Plexin B3 is required for Semaphorin signaling transduction. Our data suggest that zebrafish could be an ideal animal model for investigating the role and mechanisms of Sema3E and Plexin B3 in vivo.

UNC5C: Novel Gene Associated with Psychiatric Disorders Impacts Dysregulation of Axon Guidance Pathways.

The UNC-5 family of netrin receptor genes, predominantly expressed in brain tissues, plays a pivotal role in various neuronal processes. Mutations in genes involved in axon development contribute to a wide spectrum of human diseases, including developmental, neuropsychiatric, and neurodegenerative disorders. The NTN1/DCC signaling pathway, interacting with UNC5C, plays a crucial role in central nervous system axon guidance and has been associated with psychiatric disorders during adolescence in humans. Whole-exome sequencing analysis unveiled two compound heterozygous causative mutations within the UNC5C gene in a patient diagnosed with psychiatric disorders. In silico analysis demonstrated that neither of the observed variants affected the allosteric linkage between UNC5C and NTN1. In fact, these mutations are located within crucial cytoplasmic domains, specifically ZU5 and the region required for the netrin-mediated axon repulsion of neuronal growth cones. These domains play a critical role in forming the supramodular protein structure and directly interact with microtubules, thereby ensuring the functionality of the axon repulsion process. We emphasize that these mutations disrupt the aforementioned processes, thereby associating the UNC5C gene with psychiatric disorders for the first time and expanding the number of genes related to psychiatric disorders. Further research is required to validate the correlation of the UNC5C gene with psychiatric disorders, but we suggest including it in the genetic analysis of patients with psychiatric disorders.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Beyond a Transmission Cable-New Technologies to Reveal the Richness in Axonal Electrophysiology.

The axon is a neuronal structure capable of processing, encoding, and transmitting information. This assessment contrasts with a limiting, but deeply rooted, perspective where the axon functions solely as a transmission cable of somatodendritic activity, sending signals in the form of stereotypical action potentials. This perspective arose, at least partially, because of the technical difficulties in probing axons: their extreme length-to-diameter ratio and intricate growth paths preclude the study of their dynamics through traditional techniques. Recent findings are challenging this view and revealing a much larger repertoire of axonal computations. Axons display complex signaling processes and structure-function relationships, which can be modulated via diverse activity-dependent mechanisms. Additionally, axons can exhibit patterns of activity that are dramatically different from those of their corresponding soma. Not surprisingly, many of these recent discoveries have been driven by novel technology developments, which allow for in vitro axon electrophysiology with unprecedented spatiotemporal resolution and signal-to-noise ratio. In this review, we outline the state-of-the-art in vitro toolset for axonal electrophysiology and summarize the recent discoveries in axon function it has enabled. We also review the increasing repertoire of microtechnologies for controlling axon guidance which, in combination with the available cutting-edge electrophysiology and imaging approaches, have the potential for more controlled and high-throughput in vitro studies. We anticipate that a larger adoption of these new technologies by the neuroscience community will drive a new era of experimental opportunities in the study of axon physiology and consequently, neuronal function.

Tissue-Targeted Transcriptomics Reveals SEMA3D Control of Hypoglossal Nerve Projection to Mouse Tongue Primordia.

The mouse hypoglossal nerve originates in the occipital motor nuclei at embryonic day (E)10.5 and projects a long distance, reaching the vicinity of the tongue primordia, the lateral lingual swellings, at E11.5. However, the details of how the hypoglossal nerve correctly projects to the primordia are poorly understood. To investigate the molecular basis of hypoglossal nerve elongation, we used a novel transcriptomic approach using the ROKU method. The ROKU algorithm identified 3825 genes specific for lateral lingual swellings at E11.5, of which 34 genes were predicted to be involved in axon guidance. Ingenuity Pathway Analysis-assisted enrichment revealed activation of the semaphorin signaling pathway during tongue development, and quantitative PCR showed that the expressions of Sema3d and Nrp1 in this pathway peaked at E11.5. Immunohistochemistry detected NRP1 in the hypoglossal nerve and SEMA3D as tiny granules in the extracellular space beneath the epithelium of the tongue primordia and in lateral and anterior regions of the mandibular arch. Fewer SEMA3D granules were localized around hypoglossal nerve axons and in the space where they elongated. In developing tongue primordia, tissue-specific regulation of SEMA3D might control the route of hypoglossal nerve projection via its repulsive effect on NRP1.

Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

Stromal Cell-SLIT3/Cardiomyocyte-ROBO1 Axis Regulates Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND: Recently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress. METHODS: We performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling. RESULTS: We first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction. CONCLUSIONS: Collectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Chromatin remodeling protein CHD4 regulates axon guidance of spiral ganglion neurons in developing cochlea.

The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler. De-novo pathogenic variants of CHD4 cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients with SIHIWES show delayed development, intellectual disability, facial dysmorphism, and hearing loss. Many cochlear cell types, including spiral ganglion neurons (SGNs), express CHD4. SGNs are the primary afferent neurons that convey sound information from the cochlea, but the function of CHD4 in SGNs is unknown. We employed the Neurog1(Ngn1) CreERT2 Chd4 conditional knockout animals to delete Chd4 in SGNs. SGNs are classified as type I and type II neurons. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and ephrin receptor signaling pathway. We validated increased Epha4 transcripts in SGNs from Chd4 conditional knockout cochleae. The results suggest that CHD4 attenuates the transcription of axon guidance genes to form the stereotypic pattern of SGN peripheral projections. The results implicate epigenetic changes in circuit wiring by modulating axon guidance molecule expression and provide insights into neurodevelopmental diseases.