Embryonic Dorsal Root Ganglion Neurons Research Articles

Dental Pulp Stem Cell-Derived Exosomes Promote Sciatic Nerve Regeneration via Optimizing Schwann Cell Function.

Repair strategies for injured peripheral nerve have achieved great progresses in recent years. However, the clinical outcomes remain unsatisfactory. Recent studies have found that exosomes secreted by dental pulp stem cells (DPSC-exos) have great potential for applications in nerve repair. In this study, we evaluated the effects of human DPSC-exos on improving peripheral nerve regeneration. Initially, we established a coculture system between DPSCs and Schwann cells (SCs) in vitro to assess the effect of DPSC-exos on the activity of embryonic dorsal root ganglion neurons (DRGs) growth in SCs. We extracted and labeled human DPSC-exos, which were subsequently utilized in uptake experiments in DRGs and SCs. Subsequently, we established a rat sciatic nerve injury model to evaluate the therapeutic potential of DPSC-exos in repairing sciatic nerve damage. Our findings revealed that DPSC-exos significantly promoted neurite elongation by enhancing the proliferation, migration, and secretion of neurotrophic factors by SCs. In vivo, DPSC-exos administration significantly improved the walking behavior, axon regeneration, and myelination in rats with sciatic nerve injuries. Our study underscores the vast potential of DPSC-exos as a therapeutic tool for tissue-engineered nerve construction.

Axonal Transport Defect in Gigaxonin Deficiency Rescued by Tubastatin A

SummaryGiant axonal neuropathy (GAN) is a disease caused by a deficiency of gigaxonin, a mediator of the degradation of intermediate filament (IF) proteins. A lack of gigaxonin alters the turnover of IF proteins, provoking accumulation and disorganization of neurofilaments (NFs) in neurons, a hallmark of the disease. However, the effects of IF disorganization on neuronal function remain unknown. Here, we report that cultured embryonic dorsal root ganglia (DRG) neurons derived from Gan−/− mice exhibit accumulations of IF proteins and defects in fast axonal transport of organelles. Kymographs generated by time-lapse microscopy revealed substantial reduction of anterograde movements of mitochondria and lysosomes in axons of Gan−/− DRG neurons. Treatment of Gan−/− DRG neurons with Tubastatin A (TubA) increased the levels of acetylated tubulin and it restored the normal axonal transport of these organelles. Furthermore, we tested the effects of TubA in a new mouse model of GAN consisting of Gan−/− mice with overexpression of peripherin (Prph) transgene. Treatment of 12-month-old Gan−/−;TgPer mice with TubA led to a slight amelioration of motor function, especially a significant improvement of gait performance as measured by footprint analyses. Moreover, TubA treatment reduced the abnormal accumulations of Prph and NF proteins in spinal neurons and it boosted the levels of Prph transported into peripheral nerve axons. These results suggest that drug inhibitors of histone deacetylase aiming to enhance axonal transport should be considered as a potential treatment for GAN disease.

Coactivator-associated arginine methyltransferase 1 controls oligodendrocyte differentiation in the corpus callosum during early brain development.

Protein arginine methylation has been recognized as one of key posttranslational modifications for refined protein functions, mediated by protein arginine methyltransferases (Prmts). Coactivator-associated arginine methyltransferase (Carm1, also known as Prmt4) participates in various cellular events, such as cell survival, proliferation, and differentiation through its protein arginine methylation activities. Carm1 regulates cell proliferation of a neuronal cell line and is reportedly expressed in the mammalian brain. However, its detailed function in the central nervous system, particularly in glial cells, remains largely unexplored. In this study, Carm1 exhibited relatively high expression in oligodendrocyte (OL) lineage cells present in the corpus callosum of the developing brain, followed by a remarkable downregulation after active myelination. The suppression of Carm1 activity by inhibitors in isolated oligodendrocyte precursor cells (OPCs) reduced the number of Ki67-expressing and BrdU-incorporated proliferating cells. Furthermore, Carm1 inactivation attenuated OL differentiation, as determined by the expression of Plp, a reliable myelin-related marker. It also impaired the extension of OL processes, accompanied by a significant reduction in gene expression related to OL differentiation and myelination, such as Sox10, Cnp, Myrf, and Mbp. In addition, OLs co-cultured with embryonic dorsal root ganglia neurons demonstrated that Carm1 activity is required for the appropriate formation of myelin processes and myelin sheaths around neuronal axons, and the induction of the clustering of Caspr, a node of Ranvier structural molecule. Thus, we propose that Carm1 is an essential molecule for the development of OPCs and OLs during brain development.

Adrenomedullin increases cAMP accumulation and BDNF expression in rat DRG and spinal motor neurons.

Objective(s):Adrenomedullin (AM) has high expression in the spinal cord. In this study, we investigated the expression of AM and its receptor components, including calcitonin receptor-like receptor (CLR) and receptor activity modifying proteins (RAMPs) in dorsal root ganglion (DRG) and spinal motor (SM) neurons. Furthermore, the effects of AM on cAMP/cAMP response element-binding protein (CREB), AKT/glycogen synthase kinase-3 beta (GSK-3β) signaling pathways, and expressions of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were evaluated. Materials and Methods:Rat embryonic DRG and SM neurons were isolated, purified, and cultured. Real-time PCR was used to assess expressions of AM, CLR, and RAMPs. cAMP levels, p-CREB, BDNF, and NT-3 were determined using an enzyme-linked immunosorbent assay. p-AKT and p-GSK-3β levels were determined by western blotting. Real-time PCR showed expressions of AM, CLR, RAMP2, and RAMP3 in both DRG and SM neurons. Results:AM increased cAMP accumulation and p-CREB levels in DRG and SM neurons. AM increased p-AKT and p-GSK-3β in DRG, but not SM neurons. AM significantly increased BDNF expression in both DRG and SM neurons. There was also an increase in NT-3 level in both DRG and SM neurons, which is statistically significant in SM neurons. Conclusion:These results showed both DRG and SM neurons are targets of AM actions in the spinal cord. An increase in BDNF expression by AM in both DRG and SM neurons suggests the possible beneficial role of AM in protecting, survival, and regeneration of sensory and motor neurons.

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.

Adrenomedullin protects rat dorsal root ganglion neurons against doxorubicin-induced toxicity by ameliorating oxidative stress.

Objective(s):Despite effective anticancer effects, the use of doxorubicin (DOX) is hindered due to its cardio and neurotoxicity. The neuroprotective effect of adrenomedullin (AM) was shown in several studies. The present study aimed to evaluate the possible protective effects of AM against DOX-induced toxicity in dorsal root ganglia (DRGs) neurons. Materials and Methods:Rat embryonic DRG neurons were isolated and cultured. The effect of various concentrations of DOX (0.0 to 100 µM) in the absence or presence of AM (3.125 -100 nM) on cell death, apoptosis, oxidative stress, expression of tumor necrosis-α (TNF-α), interleukin1- β (IL-1β), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP) 3 and 13, and SRY-related protein 9 (SOX9) were examined. Results:Based on MTT assay data, DOX decreased the viability of DRG neurons in a dose and time-dependent manner (IC50=6.88 µm) while dose-dependently, AM protected DRG neurons against DOX-induced cell death. Furthermore, results of annexin V apoptosis assay revealed the protective effects of AM (25 nm) against DOX (6.88 µM)-induced apoptosis and necrosis of DRG neurons. Also, AM significantly ameliorated DOX-induced oxidative stress in DRG neurons. Real-time PCR results showed a significant increase in the expression of TNF-α, IL-1β, iNOS, MMP 3, and MMP 13, and a decrease in the expression of SOX9 following treatment with DOX. Treatment with AM (25 nM) significantly reversed the effects of DOX on the above-mentioned genes expression.Conclusion:Our findings suggest that AM can be considered a novel ameliorating drug against DOX-induced neurotoxicity.

Mechano growth factor interacts with nucleolin to protect against cisplatin-induced neurotoxicity

Mechano growth factor (MGF) is an alternatively spliced form of insulin-like growth factor-1 (IGF-1) that has shown to be neuroprotective against 6-hydroxydopamine toxicity and ischemic injury in the brain. MGF also induces neural stem cell proliferation in the hippocampus and preserves olfactory function in aging mice. Cisplatin is a chemotherapy drug that induces peripheral neuropathy in 30–40% of treated patients. Our studies were designed to see if MGF would protect dorsal root ganglion (DRG) neurons from cisplatin-induced neurotoxicity and to identify potential mechanisms that may be involved. Expression of endogenous MGF in adult DRG neurons in vivo ameliorated cisplatin-induced thermal hyperalgesia. Exogenous MGF and MGF with a cysteine added to the N-terminus (CMGF) also protected embryonic DRG neurons from cisplatin-induced cell death in vitro. Mass spectroscopy analysis of proteins bound to MGF showed that nucleolin is a key-binding partner. Antibodies against nucleolin prevented the neuroprotective effect of MGF and CMGF in culture. Both nucleolin and MGF are located in the nucleolus of DRG neurons. RNAseq of RNA associated with MGF indicated that MGF may be involved in RNA processing, protein targeting and transcription/translation. Nucleolin is an RNA binding protein that is readily shuttled between the nucleus, cytoplasm and plasma membrane. Nucleolin and MGF may work together to prevent cisplatin-induced neurotoxicity. Exploring the known mechanisms of nucleolin may help us better understand the mechanisms of cisplatin toxicity and how MGF protects DRG neurons.

Assessing Axonal Degeneration in Embryonic Dorsal Root Ganglion Neurons In Vitro.

The molecular players regulating the axon degeneration pathway have been identified using in vitro experimental models. Here, we describe an in vitro assay to assess the axonal fragmentation induced by mechanical injury to axons in cultured mouse embryonic dorsal root ganglion (DRG) neurons. DRG neurons are pseudounipolar and therefore suitable for an assay of axonal degeneration after injury. In addition, the time course of the axonal fragmentation is stereotyped, enabling the identification of reagents that either expedite or impede the degeneration process. With an image-based quantification method, the in vitro degeneration assay can be utilized as a platform supporting high-throughput screens for pharmacological or genetic reagents delaying axon degeneration.

Serum-deprived differentiated neuroblastoma F-11 cells express functional dorsal root ganglion neuron properties.

The isolation and culture of dorsal root ganglion (DRG) neurons cause adaptive changes in the expression and regulation of ion channels, with consequences on neuronal excitability. Considering that not all neurons survive the isolation and that DRG neurons are heterogeneous, it is difficult to find the cellular subtype of interest. For this reason, researchers opt for DRG-derived immortal cell lines to investigate endogenous properties. The F-11 cell line is a hybridoma of embryonic rat DRG neurons fused with the mouse neuroblastoma line N18TG2. In the proliferative condition, F-11 cells do not display a gene expression profile correspondent with specific subclasses of sensory neurons, but the most significant differences when compared with DRGs are the reduction of voltage-gated sodium, potassium and calcium channels, and the small amounts of TRPV1 transcripts. To investigate if functional properties of mature F-11 cells showed more similarities with those of isolated DRG neurons, we differentiated them by serum deprivation. Potassium and sodium currents significantly increased with differentiation, and biophysical properties of tetrodotoxin (TTX)-sensitive currents were similar to those characterized in small DRG neurons. The analysis of the voltage-dependence of calcium currents demonstrated the lack of low threshold activated components. The exclusive expression of high threshold activated Ca2+ currents and of TTX-sensitive Na+ currents correlated with the generation of a regular tonic electrical activity, which was recorded in the majority of the cells (80%) and was closely related to the activity of afferent TTX-sensitive A fibers of the proximal urethra and the bladder. Responses to capsaicin and substance P were also recorded in ~20% and ~80% of cells, respectively. The percentage of cells responsive to acetylcholine was consistent with the percentage referred for rat DRG primary neurons and cell electrical activity was modified by activation of non-NMDA receptors as for embryonic DRG neurons. These properties and the algesic profile (responses to pH5 and sensitivity to both ATP and capsaicin), proposed in literature to define a sub-classification of acutely dissociated rat DRG neurons, suggest that differentiated F-11 cells express receptors and ion channels that are also present in sensory neurons.

Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons.

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.

Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx.

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro. We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca2+, and here, we address the mechanism of Ca2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca2+ influx in sensory axon degeneration.

Creating Biomimetic Anisotropic Architectures with Co-Aligned Nanofibers and Macrochannels by Manipulating Ice Crystallization.

The continuous evolution of tissue engineering scaffolds has been driven by the desire to recapitulate structural features and functions of the natural extracellular matrix (ECM). However, it is still an extreme challenge to create a three-dimensional (3D) scaffold with both aligned nanofibers and aligned interconnected macrochannels to mimic the ECM of anisotropic tissues. Here, we develop a facile strategy to create such a scaffold composed of oriented nanofibers and interconnected macrochannels in the same direction, with various natural polymers typically used for tissue regeneration. The orientation of nanofibers and interconnected macrochannels can be easily tuned by manipulating ice crystallization. The scaffold demonstrates both structural and functional features similar to the natural ECM of anisotropic tissues. Taking silk fibroin as an example, the scaffold with radially oriented nanofibers and interconnected macrochannels is more efficient for capturing cells and promoting the growth of both nonadherent embryonic dorsal root ganglion neurons (DRGs) and adherent human umbilical vein endothelial cells (HUVECs) compared to the widely used scaffold types. Interestingly, DRGs and neurites on the SF scaffold demonstrate a 3D growth mode similar to that of natural nerve tissues. Furthermore, the coaligned nanofibers and macrochannels of the scaffold can direct HUVECs to assemble into blood vessel-like structures and their collagen deposition in their arrangement direction. The strategy could inspire the design and development of multifunctional 3D scaffolds with desirable structural features for engineering different tissues.

Polycomb protein family member CBX7 regulates intrinsic axon growth and regeneration

Neurons in the central nervous system (CNS) lose their intrinsic ability and fail to regenerate, but the underlying mechanisms are largely unknown. Polycomb group (PcG) proteins, which include PRC1 and PRC2 complexes function as gene repressors and are involved in many biological processes. Here we report that PRC1 components (polycomb chromobox (CBX) 2, 7, and 8) are novel regulators of axon growth and regeneration. Especially, knockdown of CBX7 in either embryonic cortical neurons or adult dorsal root ganglion (DRG) neurons enhances their axon growth ability. Two important transcription factors GATA4 and SOX11 are functional downstream targets of CBX7 in controlling axon regeneration. Moreover, knockdown of GATA4 or SOX11 in cultured DRG neurons inhibits axon regeneration response from CBX7 downregulation in DRG neurons. These findings suggest that targeting CBX signaling pathway may be a novel approach for promoting the intrinsic regenerative capacity of damaged CNS neurons.

Directed Differentiation of Human Bone Marrow Stromal Cells to Fate-Committed Schwann Cells.

SummaryOur ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury.

Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3

A cyclic GMP (cGMP) signaling pathway, comprising C-type natriuretic peptide (CNP), its guanylate cyclase receptor Npr2, and cGMP-dependent protein kinase I, is critical for the bifurcation of dorsal root ganglion (DRG) and cranial sensory ganglion axons when entering the mouse spinal cord and the hindbrain respectively. However, the identity and functional relevance of phosphodiesterases (PDEs) that degrade cGMP in DRG neurons are not completely understood. Here, we asked whether regulation of the intracellular cGMP concentration by PDEs modulates the branching of sensory axons. Real-time imaging of cGMP with a genetically encoded fluorescent cGMP sensor, RT-PCR screens, insitu hybridization, and immunohistology combined with the analysis of mutant mice identified PDE2A as the major enzyme for the degradation of CNP-induced cGMP in embryonic DRG neurons. Tracking of PDE2A-deficient DRG sensory axons in conjunction with cGMP measurements indicated that axon bifurcation tolerates increased cGMP concentrations. As we found that the natriuretic peptide scavenger receptor Npr3 is expressed by cells associated with dorsal roots but not in DRG neurons itself at early developmental stages, we analyzed axonal branching in the absence of Npr3. In Npr3-deficient mice, the majority of sensory axons showed normal bifurcation, but a small population of axons (13%) was unable to form T-like branches and generated turns in rostral or caudal directions only. Taken together, this study shows that sensory axon bifurcation is insensitive to increases of CNP-induced cGMP levels and Npr3 does not have an important scavenging function in this axonal system.

Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy.

Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell‐signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small‐molecule inhibitors of key signaling pathways in an 8‐day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin‐sensitive action potentials in patch‐clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact‐dependent cues to commit bone marrow‐derived Schwann cell‐like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow‐derived Schwann cells become available for autologous transplantation and remyelination therapy. Stem Cells Translational Medicine 2017;6:369–381

RAFting the rapids of axon regeneration signaling.

The functional regeneration of damaged axons and severed connections in the mature central nervous system (CNS) remains a challenging goal of neurological research. Mature CNS neurons are refractory to axon regeneration for two major reasons, one, because the activity of cell-intrinsic mechanisms that drive axon growth during development is low – and often further suppressed after an injury – and two, because certain molecules that are part of mature extracellular matrix and myelin act as strong inhibitors of axon growth. Genetic removal of growth inhibitory molecules can increase axon sprouting, but is not sufficient to enable long-range axon growth. Since axon growth is robust during early developmental stages, it has long been hypothesized that mature injured neurons may be “reprogrammed” to the earlier growth state by re-activation of the intracellular growth signaling cascades that drive axon elongation in the developing fetus.

P.1.015 Effects of neuroactive steroids on cellular bioenergetics

Neuroactive steroids synthesized from the brain or peripheral sources are called neurosteroids. Beside their common nuclear effects, they are considered to be potent neuromodulators, acting rapidly mainly in a non-genomic manner, either through allosteric regulation of ionic channels, or through membrane-bound steroid receptors. In contrast to the situation in the adult, the neurotransmitter GABA is excitatory during development and plays a trophic role, in particular inducing calcium signals necessary for the regulation of excitability and neuronal maturation. We demonstrated that the primary metabolite of progesterone (Proges), allopregnanolone (Allo), evoked a robust Ca2+ influx in foetal hypothalamic neurons and in postnatal supraoptic nucleus (SON) neurons. In the latter, this led to oxytocin and arginine vasopressin release. Interestingly, these responses were GABAA and oxytocin-receptor-dependent. Allo is a well-known positive allosteric modulator of GABAA receptors. It is noteworthy that two other steroids, Proges and 17-beta-estradiol, displayed the same effect on Ca2+ and oxytocin release but to a lesser extent. Importantly, no effect was observed in adult neurons from the SON, or in neurohypophysial axon terminals, regardless of the stage. The molecular mechanisms of the neurosteroid actions are multifaceted and depend on the type of cells, and are thus extremely interesting and challenging. In the peripheral nervous system, Allo and Proges surprisingly inhibited the GABA-induced Ca2+ increases in embryonic dorsal root ganglion neurons. We propose that this rapid, reversible and dose-dependent phenomenon (at very low concentrations) was mediated by membrane Proges receptors, since transcripts for a newly discovered receptor protein, 25-Dx, were detected in our model. Recently, novel families of membrane steroid receptors, activating intracellular-signalling pathways such as MAP kinases, have been identified and described. This opens new perspectives to understand the intracellular machinery involved in the interaction between neuropeptides and neurosteroids, two major regulators of hypothalamo-neurohypophysial system development.

In vitro anticancer activity and neurotoxicity of novel heavy metal-based anticancer complexes

Cisplatin is one of the most effective metal-based anticancer agents, targeting a large number of solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. In this work we investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to affect the proteasome machinery in DRG neurons. The copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4] PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. Furthermore, based on the content in heavy metal-based atoms, the complexes used in this study are suitable candidates for Photon Activation Therapy (PAT). Pretreatment of IGROV-1 cells with [Cu(PTA)4]PF6 induces an increase cell death with respect to drug or synchrotron (SR) alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment.

Trophic factor and hormonal regulation of neurite outgrowth in sensory neuron-like 50B11 cells

Sensory axon integrity and regenerative capacity are important considerations in understanding neuropathological conditions characterized by hyper- or insensitivity. However, our knowledge of mechanisms regulating axon outgrowth are limited by an absence of suitable high-throughput assay systems. The 50B11 cell line generated from rat embryonic dorsal root ganglion neurons offers a promising model for screening assays. Prior characterization shows that these cells express cytoskeletal proteins and genes encoding ion channels and neurotrophin receptors in common with sensory nociceptor neurons. In the present study we further characterized 50B11 cells in regard to their phenotypes and responsiveness to neurotrophic and hormonal factors. 50B11 cells express neuronal cytoplasmic proteins including beta-3 tubulin, peripherin (a marker of unmyelinated neurons), and the pan-neuronal ubiquitin hydrolase, PGP9.5. Only PGP9.5 immunoreactivity was uniformly distributed throughout soma and axons, and therefore presents the best means for visualizing the entire axon arbor. All cells co-express both NGF and GDNF receptors and addition of ligands increased neurite length. 50B11 cells also showed immunoreactivity for the estrogen receptor-α and the angiotensin receptor type II, and both 17-β estradiol and angiotensin II increased outgrowth by differentiated cells. 50B11 cells therefore show features reported previously for primary unmyelinated nociceptor neurons, including responsiveness to classical neurotrophins and hormonal modulators. Coupled with their ease of culture and predictable differentiation, 50B11 cells represent a promising cell line on which to base assays that more clearly reveal mechanisms regulating axon outgrowth and integrity.