A comparison between antisense P75NTR oligonucleotides and neurotrophic factors in promoting the survival of postnatal sensory neurons in vitro

Summary The 75-kDa low-affinity neurotrophin receptor (p75^NTR) has been shown in previous reports to mediate neuronal cell death in vitro and in vivo under certain circumstances. Antisense oligonucleotides directed against p75^NTR promote the survival of nerve growth factor-deprived dorsal root ganglia sensory neurons in vitro (Barrett, G.; Bartlett, P., Proc. Natl. Acad. Sci. USA 91:6501–6505; 1994) and axotomized dorsal root ganglia sensory neurons in vivo (Cheema, S. S.; Barrett, G. L.; Bartlett, P. F., J. Neurosci. Res. 46:239–245; 1996). In this study we compared the neuroprotective effects of antisense p75^NTR oligonucleotides with two neurotrophic factors, namely nerve growth factor (NGF) and leukemia inhibitory factor, on cultured sensory neurons derived from postnatal day 7 and 14 rat dorsal root ganglia. After 3 d in culture, treatment with the neurotrophic factors had significant survival effects on sensory neuron cultures compared to treatment with basal medium (control). However, after 6 and 9 d in culture these rescue effects were not apparent. In contrast, antisense p75^NTR oligonucleotides rescued significantly higher numbers of dorsal root ganglia sensory neurons after 6 and 9 d in culture than treatment with neurotrophic factors, sense oligonucleotides, and basal medium. Furthermore, antisense p75^NTR oligonucleotides rescued trkA-, B-, and C-expressing neurons, while NGF and leukemia inhibitory factor targeted primarily the trkA-positive neurons. These findings suggest that antisense-based strategies that inhibit gene expression of cytotoxic molecules are more efficient at preventing postnatal sensory neuronal death in vitro than treatment with individual neurotrophic factors.     

Role of Calcium Signalling in the Development of Pain Syndromes

A comparative analysis of two models of nociceptive and neuropathic pain in rodents, carrageenan peripheral inflammation of soft tissues and streptozotocin-induced diabetic neuropathy, is presented in the review. Modern concepts on the pathways of transmission of pain information are analyzed. A few aspects of possible involvement at calcium signalling in neurons in the development of pain syndromes are discussed. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 184–190, March–April, 2005.     

Effects of L1 blockade on sensory axon outgrowth and pathfinding in the chick hindlimb

In the developing chick hindlimb, sensory axons, which grow together in bundles as they extend distally, and the motoneuron axons they encounter express the cell adhesion molecule L1. Following injection of function-blocking anti-L1 antibodies into the limb at stage 25, some sensory axons choose inappropriate peripheral nerves even though motoneuron pathfinding is unaffected. Here, to further elucidate L1's role, we assessed the effects of this perturbation using pathway tracing, immune labeling, confocal microscopy, and electron microscopy. After L1 blockade, sensory axons were still bundled and closely apposed. However, clear signs of decreased adhesion were detectable ultrastructurally. Further, sensory axons grew into the limb more slowly than normal, wandering more widely, branching more frequently, and sometimes extending along inappropriate peripheral nerves. Sensory axons that ultimately projected along different cutaneous nerves showed increased intermixing in the spinal nerves, due to errors in pathfinding and also to a decreased ability to segregate into nerve-specific fascicles. These results suggest that, in the highly complex in vivo environment, as in tissue culture, L1 stimulates axon growth and enhances fasciculation, and that these processes contribute to the orderly, timely, and specific growth of sensory axons into the limb.     

Axotomy-induced ornithine decarboxylase activity in the mouse dorsal root ganglion is inhibited by the vinca alkaloids

Vinca alkaloids were used to study the role of retrograde axon transport (RT) in activating neuron perikaryal repair response to nerve transection. Mouse lumbar dorsal root ganglia (DRG) (L₄-L₆) were excised 48 hours after unilateral transection of the sciatic nerve and ornithine decarboxylase (ODC) activity determined. ODC activity in DRG ipsilateral to nerve transection was increased 10–20 fold over contralateral values. Typical ODC activities in ipsilateral and contralateral DRG samples were 6.18±1.4 and 0.31±0.09 pmol¹⁴CO₂ released/h/3DRG, respectively. Systemic administration of single doses of either vincristine (1 mg/kg) or vinblastine (5 mg/kg) immediately prior to axotomy attenuated ODC induction in ipsilateral DRG by 39% and 47%, respectively. A direct inhibition of ODC activity in the DRG appears unlikely since only high concentrations of vinblastine (0.5–1.0 mM) were able to inhibit ODC activity in vitro. We suggest vinca alkaloids inhibit ODC induction as a consequence of distupting retrograde axonal transport. Interruption of this intracellular communication mechanism may be etiologically linked to the distal axon degeneration which follows repetitive exposure to vinca alkaloids and other agents that induce toxic axonal neuropathy.     

实验性结肠炎对脊髓传入神经元中降钙素基因相关肽和香草酸受体1表达的调节

本文旨在探讨肠道局部炎症对脊髓肠道感觉传入神经通路的近期及远期效应,应用三硝基苯磺酸(trinitrobenzenesulfonic acid,TNBS)建立大鼠结肠炎动物模型,用DiI(3)逆行神经标记法识别支配肠道炎症部位的脊髓背根神经节(dorsalrootganglia,DRG)神经元,通过肉眼观察、平均组织损伤评分及髓过氧化物酶活性测定等方法评价肠道组织的炎症反应状态,用免疫组织化学法测定香草酸受体l(vanilloid receptor 1,VRl)和降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在支配结肠炎症部位的DRG神经元中的表达,比较炎症不同阶段(给予TNBS后7、21、42 d)CGRP和VRI阳性神经元的数目.结果显示,炎症急性期(即给予TNBS后7 d)结肠黏膜肉眼可见明显损伤,同时相应DRG中表达CGRP及VRl的神经元增加近2倍[(95.38±9.45)%VS(42.86±5.02)%,(89.23±8.21)%VS(32.54±4.58)%].给予TNBS后21、42 d,肠道炎症反应已完全消退,但表达CGRP及VRl的DRG神经元数目仍明显高于对照组[(86.25±8.21)%,(68.28±7.12)%VS(42.86±5.02)%;(67.22±6.52)%,(56.25±4.86)%VS(32.54±4.58)%].结果提示,肠道局部炎症可以上调支配肠道的脊髓传入神经元中CGRP和VRl的表达,这种异常表达可以持续至肠道炎症反应消退后的一定时间.     

The tetrodotoxin-insensitive sodium current in rat dorsal root ganglia is unlikely to involve the expression of the tetrodotoxin-resistant sodium channel,SkM2

Tetrodotoxin-insensitive (TTX-I) sodium currents have been recorded from newborn and adult rat sensory neurons, but the sodium channel gene(s) responsible for the TTX-I current are unknown. Because SkM2, one of six voltage-sensitive sodium channel genes cloned from rat, encodes the only cloned channel that is relatively resistant to tetrodotoxin, we sought to test whether the TTX-I current in rat sensory neurons is due to the SkM2 channel. We hypothesized that the TTX-I current might be generated from (1) an RNA splicing variant of SkM2, (2) post-translational modification of the SkM2 protein, or (3) interaction with altenate additional channel subunits. SkM2 mRNA expression was examined in newborn rat dorsal root ganglia (DRG) by RNase arotection assay. No SkM2 expression was detected. Therefore, we conclude that the TTX-I sodium current in DRG is unlikely to result from the expression of the SkM2 gene.     

Abundance of triacylglycerols in ganglia and their depletion in diabetic mice: implications for the role of altered triacylglycerols in diabetic neuropathy

Herein, we report the first study on the mass distribution and molecular species composition of abundant triacylglycerols (TAG) in ganglia. This study demonstrates five novel findings. First, unanticipated high levels of TAG were present in all examined ganglia from multiple species (e.g. mouse, rat and rabbit). Second, ganglial TAG mass content is location-dependent. Third, the TAG mass levels in ganglia are species-specific. Fourth, dorsal root ganglial TAG mass levels in streptozotocin-induced diabetic mice are dramatically depleted relative to those found in untreated control mice. Fifth, mouse ganglial TAG mass levels decrease with age although molecular species composition is not changed. Collectively, these results indicate that TAG is an important component of ganglia and may potentially contribute to pathological alterations in peripheral neuronal function in diabetic neuropathy.     

Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord

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Sensory neurons derived from diabetic rats have diminished internal Ca2+ stores linked to impaired re-uptake by the endoplasmic reticulum

Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca²⁺ signalling. Previous work has described abnormalities in Ca²⁺ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca²⁺ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase). DRG (dorsal root ganglia) sensory neurons from age-matched normal and 3–5-month-old STZ (streptozotocin)-diabetic rats (an experimental model of Type 1 diabetes) were cultured. At 1–2 days in vitro an array of parameters were measured to investigate Ca²⁺ homoeostasis including (i) axonal levels of intracellular Ca²⁺, (ii) Ca²⁺ uptake by the ER (endoplasmic reticulum), (iii) assessment of Ca²⁺ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv) determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca²⁺ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca²⁺ into the ER was reduced by 2-fold (P<0.05) in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca²⁺ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca²⁺ homoeo<1?show=[fo]?>stasis possibly triggered by sub-optimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.     

New Aspects of Progesterone Interactions with the Actin Cytoskeleton and Neurosteroidogenesis in the Cerebellum and the Neuronal Growth Cone

The impact of progesterone on neuronal tissues in the central (CNS) and peripheral (PNS) nervous system is of significant scientific and therapeutic interest. Glial and neuronal cells of vertebrates express steroidogenic enzymes, and are able to synthesize progesterone de novo from cholesterol. Progesterone is described to have neuroprotective, neuroreparative, anti-degenerative, and anti-apoptotic effects in the CNS and the PNS. Thus, the first clinical studies promise new therapeutic options using progesterone in the treatment of patients with traumatic brain injury. Additionally, experimental data from different animal models suggest further positive effects of progesterone on neurological diseases such as cerebral ischemia, peripheral nerve injury and amyothropic lateral sclerosis. In regard to this future clinical use of progesterone, we discuss in this review the underlying physiological principles of progesterone effects in neuronal tissues. Mechanisms leading to morphological reorganizations of neurons in the CNS and PNS affected by progesterone are addressed, with special focus on the actin cytoskeleton. Furthermore, new aspects of a progesterone-dependent regulation of neurosteroidogenesis mediated by the recently described progesterone binding protein PGRMC1 in the nervous system are discussed.     

Netrin-1-independent adenosine A2b receptor activation regulates the response of axons to netrin-1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCα) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCα and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCα-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCα-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.     

Streptozotocin Stimulates the Ion Channel TRPA1 Directly: INVOLVEMENT OF PEROXYNITRITE*

Streptozotocin (STZ)-induced diabetes is the most commonly used animal model of diabetes. Here, we have demonstrated that intraplantar injections of low dose STZ evoked acute polymodal hypersensitivities in mice. These hypersensitivities were inhibited by a TRPA1 antagonist and were absent in TRPA1-null mice. In wild type mice, systemic STZ treatment (180 mg/kg) evoked a loss of cold and mechanical sensitivity within an hour of injection, which lasted for at least 10 days. In contrast, Trpa1^−/− mice developed mechanical, cold, and heat hypersensitivity 24 h after STZ. The TRPA1-dependent sensory loss produced by STZ occurs before the onset of diabetes and may thus not be readily distinguished from the similar sensory abnormalities produced by the ensuing diabetic neuropathy. In vitro, STZ activated TRPA1 in isolated sensory neurons, TRPA1 cell lines, and membrane patches. Mass spectrometry studies revealed that STZ oxidizes TRPA1 cysteines to disulfides and sulfenic acids. Furthermore, incubation of tyrosine with STZ resulted in formation of dityrosine, suggesting formation of peroxynitrite. Functional analysis of TRPA1 mutants showed that cysteine residues that were oxidized by STZ were important for TRPA1 responsiveness to STZ. Our results have identified oxidation of TRPA1 cysteine residues, most likely by peroxynitrite, as a novel mechanism of action of STZ. Direct stimulation of TRPA1 complicates the interpretation of results from STZ models of diabetic sensory neuropathy and strongly argues that more refined models of diabetic neuropathy should replace the use of STZ.     

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA^taulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA^taulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA^taulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.