Mature Dorsal Root Ganglion Neurons Research Articles

Neuroprotective effect of quercetin against radiation-induced endoplasmic reticulum stress in neurons.

The endoplasmic reticulum (ER) plays an important role in the regulation and maintenance of cellular homeostasis. However, unresolved ER stress leads to deleterious effects by inducing the accumulation of unfolded proteins in the cell. Here we have demonstrated the protective aspects of quercetin against radiation-induced ER stress and against inflammation in primary cultured dorsal root ganglion (DRG) neurons. The mature DRG neurons were pretreated with different concentrations of quercetin (5-100 μM) for 24 hours before 2 Gy gamma radiation exposure and then subjected to a cytotoxicity assay, quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that quercetin decreased the expression of BiP and C/EBP-homologous protein, the ER stress marker genes along with downregulation of tumor necrosis factor-α, JNK in irradiated DRG neurons. Furthermore, quercetin pretreatment significantly increased the cytoskeletal protein Tuj1 and the neurotrophin brain-derived neurotrophic factor in the neuron. These results indicate that quercetin plays a neuroprotective role against radiation-mediated ER stress and inflammatory responses.

Developmental downregulation of LIS1 expression limits axonal extension and allows axon pruning.

ABSTRACTThe robust axonal growth and regenerative capacities of young neurons decrease substantially with age. This developmental downregulation of axonal growth may facilitate axonal pruning and neural circuit formation but limits functional recovery following nerve damage. While external factors influencing axonal growth have been extensively investigated, relatively little is known about the intrinsic molecular changes underlying the age-dependent reduction in regeneration capacity. We report that developmental downregulation of LIS1 is responsible for the decreased axonal extension capacity of mature dorsal root ganglion (DRG) neurons. In contrast, exogenous LIS1 expression or endogenous LIS1 augmentation by calpain inhibition restored axonal extension capacity in mature DRG neurons and facilitated regeneration of the damaged sciatic nerve. The insulator protein CTCF suppressed LIS1 expression in mature DRG neurons, and this reduction resulted in excessive accumulation of phosphoactivated GSK-3β at the axon tip, causing failure of the axonal extension. Conversely, sustained LIS1 expression inhibited developmental axon pruning in the mammillary body. Thus, LIS1 regulation may coordinate the balance between axonal growth and pruning during maturation of neuronal circuits.

NKCC1 downregulation induces hyperpolarizing shift of GABA responsiveness at near term fetal stages in rat cultured dorsal root ganglion neurons.

BackgroundGABAA receptor-mediated neurotransmission is greatly influenced by cation-chloride cotransporter activity during developmental stages. In embryonic neurons Na–K–2Cl (NKCC1) cotransporters mediate active chloride uptake, thus increasing the intracellular chloride concentration associated with GABA-induced depolarization. At fetal stages near term, oxytocin-induced NKCC1 downregulation has been implicated in the developmental shift from depolarizing to hyperpolarizing GABA action. Mature dorsal root ganglion neurons (DRGN), however, express high NKCC1 levels and maintain high intracellular chloride levels with consequent GABA-induced depolarization.ResultsGramicidin-perforated patch-clamp recordings were used to assess the developmental change in chloride homeostasis in rat cultured small DRGN at the embryonic day 16 (E16) and 19 (E19). The results were compared to data previously obtained in fetal DRGN at E14 and in mature cells. A significant NKCC1 downregulation, leading to reduction in excitatory GABAergic transmission, was observed at E16 and E19.ConclusionThese results indicate that NKCC1 activity transiently decreases in DRGN at fetal stages near term. This developmental shift in GABAergic transmission may contribute to fetal analgesia and neuroprotection at birth.

A Functional Dynein–Microtubule Network Is Required for NGF Signaling Through the Rap1/MAPK Pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.

Synthesis, localization and externalization of galectin-1 in mature dorsal root ganglion neurons and Schwann cells.

We recently confirmed that oxidized galectin-1 is a novel factor enhancing axonal growth in peripheral nerves after axotomy, but the process of extracellular release and oxidization of endogenous galectin-1 in the injured nervous tissue remains unknown. In the present study, we examined the distribution of galectin-1 in adult rat dorsal root ganglia (DRG) in vivo and in vitro. By RT-PCR analysis and in situ hybridization histochemistry, galectin-1 mRNA was detected in both DRG neurons and non-neuronal cells. Immunohistochemical analyses revealed that galectin-1 was distributed diffusely throughout the cytoplasm in smaller diameter neurons and Schwann cells in DRG sections. In contrast, the immunoreactivity for galectin-1 was detected in almost all DRG neurons from an early stage in culture (3 h after seeding) and was restricted to the surface and/or extracellular region of neurons and Schwann cells at later stages in culture. In a manner similar to the primary cultured cells, we also observed the surface and extracellular expression of this molecule in immortalized adult mouse Schwann cells (IMS32). Western blot analysis has revealed that both reduced and oxidized forms of galectin-1 were detected in culture media of DRG neurons and IMS32. These findings suggest that galectin-1 is externalized from DRG neurons and Schwann cells upon axonal injury. Some of the molecules in the extracellular milieu may be converted to the oxidized form, which lacks lectin activity but could act on neural tissue as a cytokine.

Expression of neurotrophin mRNAs in the dorsal root ganglion after spinal nerve injury

Neurotrophins have specificity toward distinct subpopulations of dorsal root ganglion (DRG) neurons with different neurotrophin receptors. It has been suggested that neurotrophins also play important roles in mature DRG neurons after injury. In the present study, we examined the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) mRNAs in the DRG after a peripheral nerve injury. The data showed that following a spinal nerve ligation, the level of NGF mRNA increased 4 times over the normal level and was maintained at a high level for a period of 3 weeks. The induction of BDNF mRNA was brief (lasting less than 3 days) and lesser in quantity (∼1.7 times increase) compared to NGF expression. The expression of NT-3 mRNA was not detected either in normal or nerve injured rats. Results suggest that different neurotrophins play different functional roles in the DRG after spinal nerve injury.

Developmental Regulation of Apoptosis in Dorsal Root Ganglion Neurons

The survival of dorsal root ganglion (DRG) neurons, both in vivo and in vitro, is dependent on the availability of nerve growth factor (NGF) for a transient period early in development after which these neurons become independent of NGF for survival. The precise molecular mechanism by which developing DRG neurons gain independence from NGF has not been determined. We used an in vitro model of DRG neuronal development to test hypotheses that independence from NGF in mature DRG neurons could be caused by developmental regulation of either elements of the NGF withdrawal signal transduction pathway or of proteins important for activation of the apoptosis output pathway. Interruption of phosphotidylinositol-3 kinase activation, by treatment with the specific inhibitor LY294002, resulted in apoptosis in immature but not mature DRG neurons in a manner similar to that observed with NGF withdrawal. Further downstream along the signal transduction pathway, c-JUN phosphorylation occurred in both immature and mature DRG neurons after NGF withdrawal or treatment with LY294002, despite the fact that the older neurons did not undergo apoptosis. In contrast, the ratio of expression of the proapoptotic gene bax to antiapoptotic gene bcl-xL was many times higher in immature than mature neurons, both in vivo and in vitro. Taken together, these results strongly suggest that developmental regulation of NGF withdrawal-induced apoptosis in DRG occurs via control of the relative level of expression of members of the bcl-2 gene family.

Intracellular Calcium Levels Influence Apoptosis in Mature Sensory Neurons after Trophic Factor Deprivation

Embryonic dorsal root ganglion (DRG) neurons require nerve growth factor (NGF) for survivalin vitro.Withdrawal of NGF results in an apoptotic death in these immature DRG neurons. After time in culture, DRG neurons become progressively less dependent upon NGF for survival. Immature embryonic DRG neurons remain highly dependent upon NGF during their first 14 days in cell culture but by Day 21 the majority lose their NGF dependence for survival. During this period of maturation, the intracellular calcium concentration ([Ca2+]i) progressively increases from the lower levels found in immature DRG neurons to the higher levels that are characteristic of older or mature DRG neurons. By changing the cell culture medium to one with very low calcium, we were able to lower [Ca2+]iin the mature neurons to levels similar to those found in immature neurons. These mature neurons (e.g., E-15 DRG neurons grown for 21 days in culture), normally NGF independent, became highly dependent upon NGF for survival. The onset of DNA fragmentation is a marker of apoptotic cell death. We measured the onset of DNA fragmentation in apoptotic neurons with use of the fluorescent dye, Hoechst, in neurons maintained in either standard calcium medium (1800 mM) or in the low calcium medium (0.35 mM). A higher percentage of neurons with lowered [Ca2+]ishowed initial signs of apoptosis, i.e., DNA condensation, at earlier times after NGF deprivation. This work provides further evidence to support a “set-point” hypothesis regarding the relationship between intracellular calcium concentration and NGF dependence for survival in DRG neurons.

Axon Regeneration in Vitro on Physiologically Relevant Substrata

The study of axon growth in culture is limited by a poor understanding of the relative contribution of each of a complex array of factors, which include diffusible, axon growth-modulating molecules and substrate-bound guidance cues available to developing and regenerating neurons in vivo. With the objective of more closely mimicking in vivo conditions, one approach we have exploited employs thin cryosections of appropriate regions of unfixed nervous tissue as culture substrata for the growth of regenerating neurons. By using this technique it is possible to culture different populations of neurons on substrata in which environmental growth-modulating factors are preserved. This form of bioassay has facilitated the study of the different neurite outgrowth responses of neurons both from different sources and at different developmental ages on varying native substrata. Using this method we have demonstrated that mature dorsal root ganglion neurons (DRG) will regrow axons only on predegenerated sciatic nerve in vitro, while immature DRG extend neurites on both intact and degenerated sciatic nerve. In contrast, both mature and neonatal DRG fail to regenerate on either fully myelinated mature optic nerve or unmyelinated embryonic optic nerve. Moreover, neonatal retinal ganglion cells do not regenerate on any of these substrata.