Role In Axon Guidance Research Articles

Abstract PO3-09-05: Potential novel treatments for high-risk subsets of breast cancer in postpartum and African American women

Abstract Semaphorin 7a (SEMA7A) is a neuroimmune molecule first recognized for its roles in axonal guidance and inflammation. More recently, SEMA7A has emerged as a driver of tumor progression in multiple types of cancer including lung, glioblastoma and breast. Our studies have focused on the roles of SEMA7A in promoting tumor cell growth and survival as well as invasion and metastasis in mouse models including xenograft and syngeneic. Using patient samples and datamining we have shown that SEMA7A can predict for recurrence in women with postpartum breast cancer (PPBC), or breast cancers diagnosed within 10 years of recent childbirth, and SEMA7A mRNA expression in TCGA is highest in young and African American (AA) women with BC, who typically have poor prognosis. Additionally, our analysis of the Cancer Genome Atlas (TCGA) breast cancer (n=593) and METABRIC cohorts (n=2,136) revealed that SEMA7A is increased in invasive disease and in the top 9% and 27% of upregulated genes (p=1.09E-19&p=0.009), respectively. Additionally, ~1.2 (TCGA) and ~1.1 (METABRIC) fold increases in SEMA7A mRNA conferred significantly decreased 5-year survival rates (p=0.009&p=0.002) and SEMA7A was in the top 4% of mRNAs upregulated in patients who died within 5 years of diagnosis 20,24-26. Cumulatively, these results suggest that identifying therapeutic vulnerabilities of SEMA7A+BC, or direct targeting of SEMA7A, could improve prognosis for these high-risk populations. We have identified several targets in pre-clinical models of SEMA7A+BC that include clinically available, FDA approved drugs, as well as novel inhibitors that exhibit efficacy. In published studies, we have shown that venetoclax, a Bcl-2 inhibitor, reverses ER+SEMA7A+BC resistance to endocrine therapy in mouse models. In unpublished studies, we have extended our observations in ER+SEMA7A+BC to show that alpelisib and a novel PI3K kinase inhibitor, GCT-007, both inhibit growth of human and mouse mammary tumor cells. Furthermore, in models of ER-SEMA7A+BC where we have shown cells to be resistant to chemotherapy, we have utilized age matched cell lines from AA and CA women to dissect the downstream pro-survival signaling that is mediated by SEMA7A and we have shown that anti-PD-1 and anti-PD-L1 therapies are efficacious in reducing tumor growth in vivo. Finally, we have developed a novel monoclonal antibody-based therapy that directly targets SEMA7A signaling via pro-survival pathways and inhibits tumor growth in vivo and in vitro. Collectively our results suggest that SEMA7A+BC should be identified clinically, and alternative treatments advised for this high-risk subset of breast cancer. Citation Format: Traci Lyons, Lyndsey Crump, Rachel Steinmetz, Kelsey Kines, Heather Fairchild, Alan Elder. Potential novel treatments for high-risk subsets of breast cancer in postpartum and African American women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-09-05.

CNTN4 modulates neural elongation through interplay with APP.

The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 -/- mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer's disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF.

Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-β (TGFβ) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis.NEW & NOTEWORTHY The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-β-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis.

Fas2EB112: a tale of two chromosomes.

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

Evaluating the Role of the Endocannabinoid System in Axon Guidance: A Literature Review.

Introduction: The endocannabinoid system (ECS) mediates the actions of cannabis and has been implicated in playing critical roles in key developmental events, including axon guidance. Although several recent studies have demonstrated ECS involvement in neurodevelopment, an emphasis on its putative role in axon guidance has not been reviewed comprehensively. Objective: The purpose of this literature review is to evaluate the interrelationships between the ECS and axon guidance. Methodology: This literature review analyzes existing literature demonstrating the normal role of endocannabinoid (eCB) signaling in axon guidance, with evidence from diverse animal models. Studies were obtained from a search strategy involving terms related to the ECS and axon guidance, and cross-checking cited literature to ensure a complete evaluation. Discussion: Cannabinoid receptors, as well as eCB synthesis and degradation machinery, appear necessary for normal axon guidance during neurodevelopment. Genetic and/or pharmacological disruption of eCB signaling results in axon growth and guidance errors, implying high sensitivity to exogenous cannabinoids. Conclusion: Overall, this review highlights the intricate connections between the ECS and axon guidance in normal neurodevelopment. The mechanistic evidence discussed suggests that alterations of the ECS through genetic and pharmacological interference disrupt its normal functioning and by extension its normal role in regulating neural circuitry formation. A comprehensive understanding of this topic will be valuable in potentially uncovering the mechanisms responsible for the neurodevelopmental defects associated with pre-natal cannabis use.

Role of Axon Guidance Molecules in Ascending and Descending Paths in Spinal Cord Regeneration.

Axon guidance molecules (AGM) are critical regulators of neural development and play a vital role in guiding axons to their target regions during spinal cord development. The correct wiring of neural circuits depends on these molecules' precise expression and function. Defects in axonal pathfinding, growth cone navigation, axonal branching, and synapse formation have far-reaching implications for neuronal circuit construction and function after CNS traumas, such as spinal cord injury (SCI), which affect the expression or activity of AGM. Ascending and descending paths in the spinal cord have been found to include many AGM, including Netrins, Slits, Semaphorins (Sema), Ephrins, and their receptors. In contrast to the repulsive signals like Slits and Semaphorins, which restrict axonal growth and guide axons away from unsuitable locations, Netrins are appealing guidance cues that encourage axonal growth and guidance. Defects in motor function and sensory processing can result from changes in the expression or activity of Ephrins or their receptors, which play an essential role in axonal guidance and synaptic plasticity in the spinal cord. Herein, we highlighted the expressions, functions, and mechanisms of AGM in ascending and descending spinal cord tracts, which can help us identify novel therapeutic targets to improve axonal regeneration and functional recovery after SCI.

Recombinant Slit2 suppresses neuroinflammation and Cdc42-mediated brain infiltration of peripheral immune cells via Robo1–srGAP1 pathway in a rat model of germinal matrix hemorrhage

BackgroundGerminal matrix hemorrhage (GMH) is a devastating neonatal stroke, in which neuroinflammation is a critical pathological contributor. Slit2, a secreted extracellular matrix protein, plays a repulsive role in axon guidance and leukocyte chemotaxis via the roundabout1 (Robo1) receptor. This study aimed to explore effects of recombinant Slit2 on neuroinflammation and the underlying mechanism in a rat model of GMH.MethodsGMH was induced by stereotactically infusing 0.3 U of bacterial collagenase into the germinal matrix of 7-day-old Sprague Dawley rats. Recombinant Slit2 or its vehicle was administered intranasally at 1 h after GMH and daily for 3 consecutive days. A decoy receptor recombinant Robo1 was co-administered with recombinant Slit2 after GMH. Slit2 siRNA, srGAP1 siRNA or the scrambled sequences were administered intracerebroventricularly 24 h before GMH. Neurobehavior, brain water content, Western blotting, immunofluorescence staining and Cdc42 activity assays were performed.ResultsThe endogenous brain Slit2 and Robo1 expressions were increased after GMH. Robo1 was expressed on neuron, astrocytes and infiltrated peripheral immune cells in the brain. Endogenous Slit2 knockdown by Slit2 siRNA exacerbated brain edema and neurological deficits following GMH. Recombinant Slit2 (rSlit2) reduced neurological deficits, proinflammatory cytokines, intercellular adhesion molecules, peripheral immune cell markers, neuronal apoptosis and Cdc42 activity in the brain tissue after GMH. The anti-neuroinflammation effects were reversed by recombinant Robo1 co-administration or srGAP1 siRNA.ConclusionsRecombinant Slit2 reduced neuroinflammation and neuron apoptosis after GMH. Its anti-neuroinflammation effects by suppressing onCdc42-mediated brain peripheral immune cells infiltration was at least in part via Robo1–srGAP1 pathway. These results imply that recombinant Slit2 may have potentials as a therapeutic option for neonatal brain injuries.

Hedgehog signaling guides migration of primordial germ cells to the Drosophila somatic gonad.

In addition to inducing nonautonomous specification of cell fate in both Drosophila and vertebrates, the Hedgehog pathway guides cell migration in a variety of different tissues. Although its role in axon guidance in the vertebrate nervous system is widely recognized, its role in guiding the migratory path of primordial germ cells (PGCs) from the outside surface of the Drosophila embryo through the midgut and mesoderm to the SGPs (somatic gonadal precursors) has been controversial. Here we present new experiments demonstrating (1) that Hh produced by mesodermal cells guides PGC migration, (2) that HMG CoenzymeA reductase (Hmgcr) potentiates guidance signals emanating from the SGPs, functioning upstream of hh and of 2 Hh pathway genes important for Hh-containing cytonemes, and (3) that factors required in Hh receiving cells in other contexts function in PGCs to help direct migration toward the SGPs. We also compare the data reported by 4 different laboratories that have studied the role of the Hh pathway in guiding PGC migration.

Role of the SLIT-ROBO signaling pathway in renal pathophysiology and various renal diseases.

SLIT ligand and its receptor ROBO were initially recognized for their role in axon guidance in central nervous system development. In recent years, as research has advanced, the role of the SLIT-ROBO signaling pathway has gradually expanded from axonal repulsion to cell migration, tumor development, angiogenesis, and bone metabolism. As a secreted protein, SLIT regulates various pathophysiological processes in the kidney, such as proinflammatory responses and fibrosis progression. Many studies have shown that SLIT-ROBO is extensively involved in various aspects of kidney development and maintenance of structure and function. The SLIT-ROBO signaling pathway also plays an important role in different types of kidney disease. This article reviews the advances in the study of the SLIT-ROBO pathway in various renal pathophysiological and kidney disorders and proposes new directions for further research in this field.

Roles of Fibroblast Growth Factors in the Axon Guidance.

Fibroblast growth factors (FGFs) have been widely studied by virtue of their ability to regulate many essential cellular activities, including proliferation, survival, migration, differentiation and metabolism. Recently, these molecules have emerged as the key components in forming the intricate connections within the nervous system. FGF and FGF receptor (FGFR) signaling pathways play important roles in axon guidance as axons navigate toward their synaptic targets. This review offers a current account of axonal navigation functions performed by FGFs, which operate as chemoattractants and/or chemorepellents in different circumstances. Meanwhile, detailed mechanisms behind the axon guidance process are elaborated, which are related to intracellular signaling integration and cytoskeleton dynamics.

Transcriptome profiling of a synergistic volumetric muscle loss repair strategy

Volumetric muscle loss overwhelms skeletal muscle’s ordinarily capable regenerative machinery, resulting in severe functional deficits that have defied clinical repair strategies. In this manuscript we pair the early in vivo functional response induced by differing volumetric muscle loss tissue engineering repair strategies that are broadly representative of those explored by the field (scaffold alone, cells alone, or scaffold + cells) to the transcriptomic response induced by each intervention. We demonstrate that an implant strategy comprising allogeneic decellularized skeletal muscle scaffolds seeded with autologous minced muscle cellular paste (scaffold + cells) mediates a pattern of increased expression for several genes known to play roles in axon guidance and peripheral neuroregeneration, as well as several other key genes related to inflammation, phagocytosis, and extracellular matrix regulation. The upregulation of several key genes in the presence of both implant components suggests a unique synergy between scaffolding and cells in the early period following intervention that is not seen when either scaffolds or cells are used in isolation; a finding that invites further exploration of the interactions that could have a positive impact on the treatment of volumetric muscle loss.

Srgap2 suppression ameliorates retinal ganglion cell degeneration in mice.

Slit-Robo GTPase-activating protein 2 (SRGAP2) plays important roles in axon guidance, neuronal migration, synapse formation, and nerve regeneration. However, the role of SRGAP2 in neuroretinal degenerative disease remains unclear. In this study, we found that SRGAP2 protein was first expressed in the retina of normal mice at the embryonic stage and was mainly located in the mature retinal ganglion cell layer and the inner nuclear layer. SRGAP2 protein in the retina and optic nerve increased after optic nerve crush. Then, we established a heterozygous knockout (Srgap2+/-) mouse model of optic nerve crush and found that Srgap2 suppression increased retinal ganglion cell survival, lowered intraocular pressure, inhibited glial cell activation, and partially restored retinal function. In vitro experiments showed that Srgap2 suppression activated the mammalian target of rapamycin signaling pathway. RNA sequencing results showed that the expression of small heat shock protein genes (Cryaa, Cryba4, and Crygs) related to optic nerve injury were upregulated in the retina of Srgap2+/- mice. These results suggest that Srgap2 suppression reduced the robust activation of glial cells, activated the mammalian target of rapamycin signaling pathway related to nerve protein, increased the expression of small heat shock protein genes, inhibited the degeneration of retinal ganglion cells, and partially restored optic nerve function.

Sema6D Promotes TREM2‐dependent Phagocytosis in Human iPSC‐derived Microglia

AbstractBackgroundMicroglia mediate key functions in inflammation and phagocytic clearance central to Alzheimer’s disease (AD). Semaphorins and their receptors, plexins and neuropilins, are best known for their role in axon guidance, but emerging roles include the regulation of innate immunity. Semaphorin receptors are highly expressed in microglia, including those encoded by AD risk genes discovered through genome‐wide association studies (e.g. PLXNA4 and PLCγ2). We investigate the function of semaphorin 6D (Sema6D) and interacting receptors in human iPSC‐derived microglia (iMG).MethodWe generated TREM2 knockout (KO) human iPSCs using CRISPR/Cas9. Following differentiation to iMG, we used a proximity labeling approach to demonstrate the interaction of known Sema6D receptors (Plexin‐A1 or Plexin‐A4) with TREM2. WT and KO cells were treated with Sema6D to assay (i) phagocytosis activity via live‐cell imaging and (ii) secretion of inflammatory cytokines via multiplex immunoassays (iii) downstream signaling events.ResultWe demonstrated that Sema6D promotes phagocytic uptake of oligomeric Aβ, and human synaptosomes, as well as significantly increases secretion of several inflammatory cytokines including IL‐1β, IL‐6, TNF‐α and IL‐8. TREM2 KO abrogates Sema6D‐induced cellular changes in iMG. Plexin‐A1 and Plexin‐A4 interact with TREM2 in the intact cell.ConclusionSema6D is a novel ligand for microglial Plexin‐A1and Plexin‐A4 and likely mediates signaling via a co‐receptor complex harboring TREM2. Sema6D is a regulator of microglial phagocytosis and cytokine release in a TREM2‐dependent manner, suggesting a Sema6D‐Plexin/TREM2 pathway as a novel therapeutic target for AD.

Netrin-4: Focus on Its Role in Axon Guidance, Tissue Stability, Angiogenesis and Tumors.

Netrin-4, a member of the Netrins family, is an important secreted protein that plays a role in axonal outgrowth and migration orientation. It was initially described that Netrin-4 had a high correlation with the laminin β-chain and promoted the growth of neurites in cultured olfactory bulb explants. Subsequently, it was discovered that Netrin-4 is involved in regulating various physiological processes, including angiogenesis, the occurrence and metastasis of various tumors, and the development of the kidney and alveoli. This paper reviews the current research on Netrin-4 since its discovery and provides a theoretical basis for further research on the biological characteristics of Netrin-4. Effects of Netrin-4. Netrin-4 regulates axon guidance, angiogenesis and the development of various tumors.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae)

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407bp, while its mRNA sequence was recognized as 2115bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

Cell segregation mechanisms play essential roles during the development of the central nervous system (CNS) to support its organization into distinct compartments. The Slit protein is a secreted signal, classically considered a paracrine repellent for axonal growth through Robo receptors. However, its function in the compartmentalization of CNS is less explored. In this work, we show that Slit and Robo3 are expressed in the same neuronal population of the Drosophila optic lobe, where they are required for the correct compartmentalization of optic lobe neuropils by the action of an autocrine/paracrine mechanism. We characterize the endocytic route followed by the Slit/Robo3 complex and detected genetic interactions with genes involved in endocytosis and actin dynamics. Thus, we report that the Slit-Robo3 pathway regulates the morphogenesis of the optic lobe through an atypical autocrine/paracrine mechanism in addition to its role in axon guidance, and in association with proteins of the endocytic pathway and small GTPases.

Synaptic and functional alterations in the development of mutant huntingtin expressing hiPSC-derived neurons.

Huntington’s disease (HD) is a monogenic disease that results in a combination of motor, psychiatric, and cognitive symptoms. It is caused by a CAG trinucleotide repeat expansion in the exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant HTT protein (mHTT) with an extended polyglutamine tract (PolyQ). Severe motor symptoms are a hallmark of HD and typically appear during middle age; however, mild cognitive and personality changes often occur already during early adolescence. Wild-type HTT is a regulator of synaptic functions and plays a role in axon guidance, neurotransmitter release, and synaptic vesicle trafficking. These functions are important for proper synapse assembly during neuronal network formation. In the present study, we assessed the effect of mHTT exon1 isoform on the synaptic and functional maturation of human induced pluripotent stem cell (hiPSC)-derived neurons. We used a relatively fast-maturing hiPSC line carrying a doxycycline-inducible pro-neuronal transcription factor, (iNGN2), and generated a double transgenic line by introducing only the exon 1 of HTT, which carries the mutant CAG (mHTTEx1). The characterization of our cell lines revealed that the presence of mHTTEx1 in hiPSC-derived neurons alters the synaptic protein appearance, decreases synaptic contacts, and causes a delay in the development of a mature neuronal activity pattern, recapitulating some of the developmental alterations observed in HD models, nonetheless in a shorted time window. Our data support the notion that HD has a neurodevelopmental component and is not solely a degenerative disease.

Neuronal Glypican4 promotes mossy fiber sprouting through the mTOR pathway after pilocarpine-induced status epilepticus in mice

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies.

TDP-43 is essential for Eph receptor-class-specific spinal motor axon trajectory into the limb

During the neural circuit formation, neuronal growth cones must be guided precisely to their neuronal or muscle targets, which can be achieved by the activation of membrane-bound guidance receptors at the periphery. However, the mechanisms that regulate the temporal availability of these receptors remain largely unknown. TAR DNA binding protein-43 (TDP-43) has been proposed to bind with the mRNAs of guidance receptors, thus prompting us to investigate its role in axon guidance of the spinal lateral motor column (LMC) neurons into the limb mesenchyme. We first identified the TDP-43 expression in the LMC neurons at the stage of axons growth into the limb using in situ mRNA hybridization. The loss and gain of TDP-43 function in chick LMC neurons redirected their axon trajectory with opposite effects. In mice, a spinal motor neuron-specific TDP-43 deletion led to the misrouting of LMC axons. Further, ectopic TDP-43 expression increased EphB protein levels in LMC neurons, suggesting that TDP-43 mediates LMC pathfinding by regulating EphB expression. Finally, TDP-43 levels influenced the growth preference of LMC neurites against ephrin-B, but not Netrin-1 and Semaphorin ligands. Our results demonstrate that TDP-43 is essential for the ephrinB:EphB signaling-mediated axon trajectory selection of LMC subtypes into the limb.

Ephrin-A3 is required for tonotopic map precision and auditory functions in the mouse auditory brainstem.

Tonotopy is a prominent feature of the vertebrate auditory system and forms the basis for sound discrimination, but the molecular mechanism that underlies its formation remains largely elusive. Ephrin/Eph signaling is known to play important roles in axon guidance during topographic mapping in other sensory systems, so we investigated its possible role in the establishment of tonotopy in the mouse cochlear nucleus. We found that ephrin-A3 molecules are differentially expressed along the tonotopic axis in the cochlear nucleus during innervation. Ephrin-A3 forward signaling is sufficient to repel auditory nerve fibers in a developmental stage-dependent manner. In mice lacking ephrin-A3, the tonotopic map is degraded and isofrequency bands of neuronal activation upon pure tone exposure become imprecise in the anteroventral cochlear nucleus. Ephrin-A3 mutant mice also exhibit a delayed second wave in auditory brainstem responses upon sound stimuli and impaired detection of sound frequency changes. Our findings establish an essential role for ephrin-A3 in forming precise tonotopy in the auditory brainstem to ensure accurate sound discrimination.