SEMA4D Knockdown Attenuates β-Catenin-Dependent Tumor Progression in Colorectal Cancer

Semaphorin 4D (SEMA4D), a protein originally demonstrated to regulate the immune system and axonal growth cone collapse in the developing central nervous system, is overexpressed in various human malignancies, including colorectal cancer (CRC). This investigation was undertaken to examine the effects of SEMA4D silencing on the biological properties of the CRC cell line. SW48 cells were transfected with a siRNA-targeting SEMA4D. The mRNA expression of underlying pro- and antiapoptotic proteins including Bax, Bcl-2, P53, and caspase-3, cancer stem cell (CSC) markers, epithelial-mesenchymal transition (EMT) markers, MMP-2, and MMP-9 was examined using qRT-PCR. Further, the protein expression of E-cadherin and β-catenin was confirmed by Western blot. SW48 cell migration and MMP activity were detected using scratch and zymography analysis, respectively. Finally, the apoptosis rate was assessed via the flowcytometry test. SEMA4D knock-down was associated with a considerable suppression of in vitro cell viability, EMT-related genes, CSC markers, β-catenin signaling pathway, sphere-forming, cell migration, and MMP-2 activity as well as induction of apoptosis. This study identifies the inhibitory effects of SEMA4D gene silencing on tumor progression. Thereby, this might conclude a possible alternative to cancer therapy by targeting several prominent pathways involved in cancer through SEMA4D suppression.

Vinaxanthone inhibits Semaphorin3A induced axonal growth cone collapse in embryonic neurons but fails to block its growth promoting effects on adult neurons

Semaphorin3A is considered a classical repellent molecule for developing neurons and a potent inhibitor of regeneration after nervous system trauma. Vinaxanthone and other Sema3A inhibitors are currently being tested as possible therapeutics to promote nervous system regeneration from injury. Our previous study on Sema3A demonstrated a switch in Sema3A’s function toward induction of nerve regeneration in adult murine corneas and in culture of adult peripheral neurons. The aim of the current study is to determine the direct effects of Vinaxanthone on the Sema3A induced adult neuronal growth. We first demonstrate that Vinaxanthone maintains its anti-Sema3A activity in embryonic dorsal root ganglia neurons by inhibiting Sema3A-induced growth cone collapse. However, at concentrations approximating its IC50 Vinaxanthone treatment does not significantly inhibit neurite formation of adult peripheral neurons induced by Sema3A treatment. Furthermore, Vinaxanthone has off target effects when used at concentrations above its IC50, and inhibits neurite growth of adult neurons treated with either Sema3A or NGF. Our results suggest that Vinaxanthone’s pro-regenerative effects seen in multiple in vivo models of neuronal injury in adult animals need further investigation due to the pleiotropic effect of Sema3A on various non-neuronal cell types and the possible effect of Vinaxanthone on other neuroregenerative signals.

Lentiviral Mediated Expression of Soluble Neuropilin 1 Inhibits Semaphorin 3A-mediated Collapse Activity in Vitro

Introduction: Semaphorin 3A (Sema 3A) is a secreted protein, which plays an integral part in developing the nervous system. It has collapse activity on the growth cone of dorsal root ganglia. After the development of the nervous system, Sema 3A expression decreases. Neuropilin 1 is a membrane receptor of Sema 3A. When semaphorin binds to neuropilin 1, the recruitment of oligodendrocyte precursor cells to the demyelinated site decreases. In Multiple Sclerosis (MS), Sema 3A expression increases and inhibits oligodendrocyte precursor cell differentiation. Therefore, the remyelination of axons gets impaired. We hypothesized that the function of Sema 3A could be inhibited by neutralizing its binding to membrane NRP1. Methods: we cloned a soluble form of mouse Neuropilin 1 (msNRP1) in a lentiviral vector and expressed the recombinant protein in HEK293T cells. Then, the conditioned medium of the transduced cells was used to evaluate the effects of the msNRP1 on the inhibition of Sema 3A-induced growth cone collapse activity. Dorsal root ganglion explants of timed pregnant (E13) mice were prepared. Then, the growth cone collapse activity of Sema 3A was assessed in the presence and absence of msNRP1-containing conditioned media of transduced and non-transduced HEK293T cells. Comparisons between groups were performed by 1-way ANOVA and post hoc Tukey tests. Results: msNRP1 was successfully cloned and transduced in HEK293T cells. The supernatant of transduced cells was concentrated and evaluated for the production of msNRP1. ELISA results indicated that transduced cells secreted msNRP1. Growth cone collapse assay showed that Sema 3A activity was significantly reduced in the presence of the conditioned medium of msNRP1-transduced HEK293T cells. Conversely, a conditioned medium of non-transduced HEK293T cells could not effectively prevent Sema 3A growth cone collapse activity. Conclusion: Our results indicated that msNRP1 was successfully produced in HEK293T cells. The secreted msNRP1 effectively prevented Sema 3A collapse activity. Therefore, msNRP1 can increase remyelination in MS lesions, although more studies using animal models are required.

Nogo-A Beyond the RhoA/ROCK Pathway – Novel Components of Intracellular Nogo-A Signaling Cascades

Nogo-A is a well-characterized myelin-associated membrane protein that restricts fibre growth and the regenerative capacity of the adult central nervous system after injury. To date Nogo-A post-receptor signalling pathway research focused on the RhoA/ROCK cascade, which can lead to growth cone collapse and neurite retraction. Much less is known about continued intracellular Nogo-A signalling mediating long-term neurite outgrowth inhibition resulting from transcriptional and translational changes. Here, we propose a simple but highly reproducible in vitro assay to study Nogo-A related signaling and neurite outgrowth inhibition in general. Furthermore, we identified ERK1/2 as downstream effector of Nogo-A, partially mediating its neurite outgrowth inhibition. We describe ERK1/2 dependent changes of translational events such as elevation of RhoA levels within the growth cone, which may potentiate the cells’ responses to Nogo-A. We also observed Nogo-A dependent upregulation of the JAK/STAT pathway inhibitors SOCS3 and KLF4 and downregulation of insulin mediated phosphorylation of AKT, indicating direct negative crosstalk between Nogo-A signalling and the growth promoting JAK/STAT and AKT/mTORC1 pathways.

Developmental Regulation of Basket Interneuron Synapses and Behavior through NCAM in Mouse Prefrontal Cortex

Parvalbumin (PV)-expressing basket interneurons in the prefrontal cortex (PFC) regulate pyramidal cell firing, synchrony, and network oscillations. Yet, it is unclear how their perisomatic inputs to pyramidal neurons are integrated into neural circuitry and adjusted postnatally. Neural cell adhesion molecule NCAM is expressed in a variety of cells in the PFC and cooperates with EphrinA/EphAs to regulate inhibitory synapse density. Here, analysis of a novel parvalbumin (PV)-Cre: NCAM F/F mouse mutant revealed that NCAM functions presynaptically in PV+ basket interneurons to regulate postnatal elimination of perisomatic synapses. Mutant mice exhibited an increased density of PV+ perisomatic puncta in PFC layer 2/3, while live imaging in mutant brain slices revealed fewer puncta that were dynamically eliminated. Furthermore, EphrinA5-induced growth cone collapse in PV+ interneurons in culture depended on NCAM expression. Electrophysiological recording from layer 2/3 pyramidal cells in mutant PFC slices showed a slower rise time of inhibitory synaptic currents. PV-Cre: NCAM F/F mice exhibited impairments in working memory and social behavior that may be impacted by altered PFC circuitry. These findings suggest that the density of perisomatic synapses of PV+ basket interneurons is regulated postnatally by NCAM, likely through EphrinA-dependent elimination, which is important for appropriate PFC network function and behavior.

Abelson-induced phosphorylation of TACC3 modulates its interaction with microtubules and affects its impact on axon outgrowth and guidance

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth and guidance, and it also regulates microtubule extension into the growth cone periphery. Together, our results suggest that phosphorylation of TACC3 is a key regulatory mechanism by which TACC3 controls axon outgrowth and pathfinding decisions of neurons during embryonic development.

Abstract WP141: Cyclic-AMP Induces Nogo-A Receptor NgR1 Internalization and Inhibits Nogo-A-Mediated Collapse of Growth Cone

Recovery of stroke and neuronal injuries requires the promotion of axonal regeneration from the remaining neurons. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. C-terminal domain of Nogo-A, Nogo-66 binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibition of neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors. In this study, by using indirect immunofluorescence and biotinylation method, we have found that a cell-permeable cAMP analog (dibutyryl-cAMP) and other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, all induced rapid internalization of the cell surface NgR1 in Neuroscreen-1 (NS-1) cells. This endocytosis of NgR1 is lipid raft mediated. These cAMP-elevating agents induced a reversible distribution of NgR1 between the cell surface and intracellular compartment; NgR1 distributed to the cell surface at low levels of cAMP and distributed to an intracellular compartment at high levels of cAMP. Using pharmacological activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA but dependent on Epac. There is a correlation between the decrease in cell surface expression of NgR1 decreased sensitivity of NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, besides axonal growth inhibitors affecting neurons, neurons by themselves self-regulate their own sensitivity to extracellular cues such as axonal growth inhibitors. This normal cellular regulatory mechanism may be therapeutically applied to overcome axonal growth inhibitors and enhance functional recovery after stroke and neuronal injuries.

Fast-diffusing p75NTR monomers support apoptosis and growth cone collapse by neurotrophin ligands

The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Inhibition of Rho GTPases in Invertebrate Growth Cones Induces a Switch in Responsiveness to Retinoic Acid

During development, growth cones are essential for axon pathfinding by sensing numerous guidance cues in their environment. Retinoic acid, the metabolite of vitamin A, is important for neurite outgrowth during vertebrate development, but may also play a role in axon guidance, though little is known of the cellular mechanisms involved. Our previous studies showed that retinoid-induced growth cone turning of invertebrate motorneurons requires local protein synthesis and calcium influx. However, the signalling pathways that link calcium influx to cytoskeletal dynamics involved in retinoid-mediated growth cone turning are not currently known. The Rho GTPases, Cdc42 and Rac, are known regulators of the growth cone cytoskeleton. Here, we demonstrated that inhibition of Cdc42 or Rac not only prevented growth cone turning toward retinoic acid but could also induce a switch in growth cone responsiveness to chemorepulsion or growth cone collapse. However, the effects of Cdc42 or Rac inhibition on growth cone responsiveness differed, depending on whether the turning was induced by the all-trans or 9-cis retinoid isomer. The effects also differed depending on whether the growth cones maintained communication with the cell body. These data strongly suggest that Cdc42 and Rac are downstream effectors of retinoic acid during growth cone guidance.