RESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Polímeros , Células Ganglionares da Retina , Humanos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Polímeros/química , Orientação de Axônios , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Propriedades de Superfície , Condutividade Elétrica , Fatores de Crescimento Neural/metabolismo , Axônios/metabolismo , Axônios/fisiologiaRESUMO
Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Íntrons , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Orientação de Axônios/genética , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Regulação da Expressão GênicaRESUMO
The axon guidance cue netrin-1 signals through its receptor DCC (deleted in colorectal cancer) to attract commissural axons to the midline. Variants in DCC are frequently associated with congenital mirror movements (CMMs). A CMM-associated variant in the cytoplasmic tail of DCC is located in a conserved motif predicted to bind to a regulator of actin dynamics called the WAVE (Wiskott-Aldrich syndrome protein-family verprolin homologous protein) regulatory complex (WRC). Here, we explored how this variant affects DCC function and may contribute to CMM. We found that a conserved WRC-interacting receptor sequence (WIRS) motif in the cytoplasmic tail of DCC mediated the interaction between DCC and the WRC. This interaction was required for netrin-1-mediated axon guidance in cultured rodent commissural neurons. Furthermore, the WIRS motif of Fra, the Drosophila DCC ortholog, was required for attractive signaling in vivo at the Drosophila midline. The CMM-associated R1343H variant of DCC, which altered the WIRS motif, prevented the DCC-WRC interaction and impaired axon guidance in cultured commissural neurons and in Drosophila. The findings reveal the WRC as a pivotal component of netrin-1-DCC signaling and uncover a molecular mechanism explaining how a human genetic variant in the cytoplasmic tail of DCC may lead to CMM.
Assuntos
Orientação de Axônios , Receptor DCC , Proteínas de Drosophila , Netrina-1 , Netrina-1/metabolismo , Netrina-1/genética , Receptor DCC/metabolismo , Receptor DCC/genética , Animais , Humanos , Orientação de Axônios/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ratos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Axônios/metabolismo , Axônios/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Camundongos , Neurônios/metabolismo , Células HEK293 , Receptores de NetrinaRESUMO
The absence or dysfunction of primary cilia, which are non-motile protrusions on cells, leads to a group of neurodevelopment disorders called ciliopathies. In a new study, Esther Stoeckli and colleagues identify the role of primary cilium-mediated sonic hedgehog (Shh) signaling in commissural axon guidance in mice and chick embryos. We caught up with first author, Alexandre Dumoulin, and corresponding author, Esther Stoeckli, Professor at the University of Zurich, to find out more about the work.
Assuntos
Cílios , Proteínas Hedgehog , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Cílios/metabolismo , Humanos , Camundongos , Embrião de Galinha , Transdução de Sinais , História do Século XXI , Orientação de Axônios , História do Século XXRESUMO
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.
Assuntos
Orientação de Axônios , Axônios , Caderinas , Protocaderinas , Células Ganglionares da Retina , Proteínas de Xenopus , Xenopus laevis , Animais , Caderinas/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Células Ganglionares da Retina/metabolismo , Xenopus laevis/embriologia , Axônios/metabolismo , Retina/metabolismo , Retina/embriologia , Vias Visuais , Técnicas de Silenciamento de Genes , Quiasma Óptico/embriologia , Quiasma Óptico/metabolismo , Colículos Superiores/embriologia , Colículos Superiores/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
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.
Assuntos
Orientação de Axônios , Axônios , Cílios , Proteínas Hedgehog , Transdução de Sinais , Cílios/metabolismo , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos , Axônios/metabolismo , Cones de Crescimento/metabolismo , Neurônios/metabolismoRESUMO
INTRODUCTION: Peripheral nerve injury (PNI) occurs in approximately 3% of all trauma patients and can be challenging to treat, particularly when injury is severe such as with a long-segmental gap. Although peripheral nerves can regenerate after injury, functional recovery is often insufficient, leading to deficits in the quality of life of patients with PNI. Although nerve autografts are the gold standard of care, there are several disadvantages to their use, namely a lack of autologous nerve material for repair. This has led to the pursuit of alternative treatment methods such as axon guidance channels (AGCs). Second-generation AGCs have been shown to be able to deliver growth-enhancing substrates for nerve repair directly to the injury site. Although our laboratory has had success with second-generation AGCs filled with Schwann cells (SCs), SCs have their own set of issues clinically. Because of this, we have begun to utilize SC-derived exosomes as an alternative, as they have the appropriate protein markers, associate to axons in high concentrations, and are able to improve nerve regeneration. However, it is unknown how SC-derived exosomes may react within second-generation AGCs; thus, the aim of the present study was to assess the ability of SC-derived exosomes to be loaded into a second-generation AGC and how they would distribute within it. MATERIALS AND METHODS: A total of 4 dry second-generation AGCs were loaded with SC-derived exosomes that were derived from green fluorescent protein (GFP)-labeled SCs. They were subsequently frozen and sliced before imaging. RESULTS: Here, we present findings that SC-derived exosomes can be loaded into second-generation AGCs through our established loading method utilizing negative pressure and are able to survive and equally distribute along the length of the AGC. CONCLUSIONS: Although only 4 second-generation AGCs were utilized, these findings indicate a potential use for SC-derived exosomes within second-generation AGCs to treat severe PNI. Future research should focus on exploring this in greater detail and in different contexts to assess the ability of SC-derived exosomes to survive at the site of injury and treat PNI.
Assuntos
Exossomos , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Células de Schwann , Células de Schwann/fisiologia , Regeneração Nervosa/fisiologia , Animais , Traumatismos dos Nervos Periféricos/terapia , Ratos , Orientação de Axônios/fisiologia , Axônios/fisiologiaRESUMO
The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 had axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that loss of Ntn1 did not affect bifurcation but rather altered turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit1, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance errors, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development.
Assuntos
Orientação de Axônios , Axônios , Gânglios Espinais , Proteínas do Tecido Nervoso , Netrina-1 , Medula Espinal , Animais , Netrina-1/metabolismo , Netrina-1/genética , Camundongos , Medula Espinal/metabolismo , Medula Espinal/embriologia , Axônios/metabolismo , Axônios/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Orientação de Axônios/fisiologia , Gânglios Espinais/metabolismo , Gânglios Espinais/embriologia , Camundongos Knockout , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genéticaRESUMO
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.
Assuntos
Orientação de Axônios , Proteínas de Drosophila , Proteoglicanas , Semaforinas , Transdução de Sinais , Animais , Semaforinas/metabolismo , Semaforinas/genética , Proteoglicanas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Axônios/metabolismo , Drosophila melanogaster/metabolismo , Glicosaminoglicanos/metabolismo , Sítios de Ligação , Ligação Proteica , Neurônios Receptores Olfatórios/metabolismoRESUMO
In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/ß and α'/ß' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/ß' neurons, controls the formation of the correct axonal pattern of α'/ß' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/ß' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/ß' neurons, which is also crucial for wiring of α'/ß' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.
Assuntos
Orientação de Axônios , Proteínas de Drosophila , Memória , Isoformas de Proteínas , Semaforinas , Animais , Axônios/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Memória/fisiologia , Metamorfose Biológica/fisiologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Semaforinas/metabolismo , Semaforinas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Ferric-tannic nanoparticles (FTs) are now considered to be new pharmaceuticals appropriate for the prevention of brain aging and related diseases. We have previously shown that FTs could activate axon guidance pathways and cellular clearance functioning in neuronal cell lines. Herein, we further investigated whether FTs could activate the two coordinated neuronal functions of axon guidance and synaptic function in rat brains and neuronal cell lines. A single intravenous injection of a safe dose of FTs has been shown to activate a protein expression of axon attractant Netrin-1 and neurotransmitter receptor GABRA4 in the cerebral cortexes of male Wistar rats. According to RNA-seq with targeted analysis, axon guidance and synapses have been enriched and Ephrin membered genes have been identified as coordinating a network of genes for such processes. In vitro, as expected, FTs are also found to activate axon guidance markers and promote neuronal tubes in neuronal cell lines. At the same time, pre-synaptic markers (synaptophysin), post-synaptic markers (synapsin), and GABRA4 neurotransmitter receptors have been found to be activated by FTs. Interestingly, synaptophysin has been found to localize along the promoted neuronal tubes, suggesting that enhanced axon guidance is associated with the formation and transportation of pre-synaptic vesicles. Preliminarily, repeated injection of FTs into adult rats every 3 days for 10 times could enhance an expression of synaptophysin in the cerebral cortex, as compared to control rats. This work demonstrates that FTs can be used for activating brain function associated with axon guidance and synaptic function.
Assuntos
Orientação de Axônios , Ratos Wistar , Sinapses , Animais , Masculino , Ratos , Sinapses/metabolismo , Encéfalo/metabolismo , Sinaptofisina/metabolismo , Compostos Férricos/metabolismo , Receptores de GABA-A/metabolismo , Netrina-1/metabolismo , Nanopartículas/química , Biomarcadores/metabolismo , Neurônios/metabolismo , Axônios/metabolismoRESUMO
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.
Assuntos
Orientação de Axônios , Proteínas Hedgehog , beta-Arrestinas , Animais , Proteínas Hedgehog/metabolismo , Ratos , Orientação de Axônios/fisiologia , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Arrestinas/genética , Feminino , Axônios/fisiologia , Axônios/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Quinases da Família src/metabolismo , Masculino , Medula Espinal/metabolismo , Medula Espinal/embriologia , Medula Espinal/citologia , Embrião de Galinha , HumanosRESUMO
Most of the patients affected by neuronopathic forms of Mucopolysaccharidosis type II (MPS II), a rare lysosomal storage disorder caused by defects in iduronate-2-sulfatase (IDS) activity, exhibit early neurological defects associated with white matter lesions and progressive behavioural abnormalities. While neuronal degeneration has been largely described in experimental models and human patients, more subtle neuronal pathogenic defects remain still underexplored. In this work, we discovered that the axon guidance receptor Deleted in Colorectal Cancer (Dcc) is significantly dysregulated in the brain of ids mutant zebrafish since embryonic stages. In addition, thanks to the establishment of neuronal-enriched primary cell cultures, we identified defective proteasomal degradation as one of the main pathways underlying Dcc upregulation in ids mutant conditions. Furthermore, ids mutant fish-derived primary neurons displayed higher levels of polyubiquitinated proteins and P62, suggesting a wider defect in protein degradation. Finally, we show that ids mutant larvae display an atypical response to anxiety-inducing stimuli, hence mimicking one of the characteristic features of MPS II patients. Our study provides an additional relevant frame to MPS II pathogenesis, supporting the concept that multiple developmental defects concur with early childhood behavioural abnormalities.
Assuntos
Iduronato Sulfatase , Mucopolissacaridose II , Doenças do Sistema Nervoso , Animais , Orientação de Axônios , Encéfalo/metabolismo , Iduronato Sulfatase/metabolismo , Mucopolissacaridose II/metabolismo , Doenças do Sistema Nervoso/patologia , Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fenótipo , Semaforina-3A , Animais , Humanos , Camundongos , Orientação de Axônios/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Semaforina-3A/metabolismo , Semaforina-3A/genética , Transdução de SinaisRESUMO
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.
Assuntos
Laringe , Regeneração Nervosa , Netrina-1 , Netrina-1/metabolismo , Animais , Humanos , Regeneração Nervosa/fisiologia , Laringe/fisiologia , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Orientação de Axônios/fisiologiaRESUMO
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.
Assuntos
Orientação de Axônios , Transtornos Mentais , Humanos , Orientação de Axônios/genética , Netrina-1/genética , Netrina-1/metabolismo , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Axônios/metabolismo , Transtornos Mentais/metabolismoRESUMO
The idea of guidance toward a target is central to axon pathfinding and brain wiring in general. In this work, we show how several thousand axonal growth cones self-pattern without target-dependent guidance during neural superposition wiring in Drosophila. Ablation of all target lamina neurons or loss of target adhesion prevents the stabilization but not the development of the pattern. Intravital imaging at the spatiotemporal resolution of growth cone dynamics in intact pupae and data-driven dynamics simulations reveal a mechanism by which >30,000 filopodia do not explore potential targets, but instead simultaneously generate and navigate a dynamic filopodial meshwork that steers growth directions. Hence, a guidance mechanism can emerge from the interactions of the axons being guided, suggesting self-organization as a more general feature of brain wiring.
Assuntos
Orientação de Axônios , Drosophila melanogaster , Cones de Crescimento , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Pseudópodes/fisiologiaRESUMO
EphB1 is required for proper guidance of cortical axon projections during brain development, but how EphB1 regulates this process remains unclear. We show here that EphB1 conditional knockout (cKO) in GABAergic cells (Vgat-Cre), but not in cortical excitatory neurons (Emx1-Cre), reproduced the cortical axon guidance defects observed in global EphB1 KO mice. Interestingly, in EphB1 cKOVgat mice, the misguided axon bundles contained co-mingled striatal GABAergic and somatosensory cortical glutamatergic axons. In wild-type mice, somatosensory axons also co-fasciculated with striatal axons, notably in the globus pallidus, suggesting that a subset of glutamatergic cortical axons normally follows long-range GABAergic axons to reach their targets. Surprisingly, the ectopic axons in EphB1 KO mice were juxtaposed to major blood vessels. However, conditional loss of EphB1 in endothelial cells (Tie2-Cre) did not produce the axon guidance defects, suggesting that EphB1 in GABAergic neurons normally promotes avoidance of these ectopic axons from the developing brain vasculature. Together, our data reveal a new role for EphB1 in GABAergic neurons to influence proper cortical glutamatergic axon guidance during brain development.
Assuntos
Orientação de Axônios , Células Endoteliais , Animais , Camundongos , Axônios/fisiologia , Neurônios GABAérgicos , Camundongos Knockout , Receptores Proteína Tirosina Quinases , Receptor EphB1/metabolismoRESUMO
Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.
Assuntos
Citosina , Dioxigenases , Animais , Citosina/metabolismo , Drosophila/genética , Drosophila/metabolismo , Metilação de DNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Orientação de Axônios , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina/metabolismo , DNA/metabolismo , Dioxigenases/genéticaRESUMO
Effective axon regeneration within the central nervous system (CNS) is pivotal for achieving functional recovery following spinal cord injury (SCI). Numerous extrinsic and intrinsic factors exert influences on the axon regeneration. While prior studies have demonstrated crucial involvement of specific members the Rab protein family in axon regeneration in the peripheral nervous system (PNS), the precise function of Rab11 in CNS axon regeneration in vivo remains elusive. Thus, our study aimed to elucidate the impact of Rab11 on the axon regeneration of Mauthner cells (M-cells) in zebrafish larvae. Our findings demonstrated that overexpression of Rab11bb via single-cell electroporation significantly promoted axon regeneration in individual M-cells. Conversely, knockdown of Rab11bb inhibited the axon regeneration of M-cells. RNA-seq analysis revealed an upregulation of ntng2b following Rab11bb overexpression. As we hypothesized, overexpression of Ntng2b markedly enhanced axon regeneration, while Ntng2b knockdown in the context of Rab11bb pro-regeneration substantially hindered axon regrowth. In conclusion, our study demonstrated that Rab11 promotes axon regeneration of single M-cell in the CNS through the Rab11/axon guidance/Ntng2b pathway.