Ryousuke Fujita

Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling.

Lysophosphatidic acid (LPA) is a bioactive lipid with activity in the nervous system mediated by G-protein-coupled receptors. Here, we examined the role of LPA signaling in the development of neuropathic pain by pharmacological and genetic approaches, including the use of mice lacking the LPA1 receptor. Wild-type animals with nerve injury develop behavioral allodynia and hyperalgesia paralleled by demyelination in the dorsal root and increased expression of both the protein kinase C γ-isoform within the spinal cord dorsal horn and the α2δ1 calcium channel subunit in dorsal root ganglia. Intrathecal injection of LPA induced behavioral, morphological and biochemical changes similar to those observed after nerve ligation. In contrast, mice lacking a single LPA receptor (LPA1, also known as EDG2) that activates the Rho–Rho kinase pathway do not develop signs of neuropathic pain after peripheral nerve injury. Inhibitors of Rho and Rho kinase also prevented these signs of neuropathic pain. These results imply that receptor-mediated LPA signaling is crucial in the initiation of neuropathic pain.

LPA-mediated demyelination in ex vivo culture of dorsal root

Lysophosphatidic acid (LPA) causes neuropathic pain with demyelination in sensory fibers. In dorsal root (DR) ex vivo culture, the addition of 0.1 microM LPA caused a characteristic demyelination at 24h in scanning and transmission electron microscopy analyses. Moreover, direct contact between C-fibers due to loss of partition by Schwann cell in Remak bundles was observed. LPA-induced demyelination of DR was concentration-dependent in the range between 0.01 and 1M, and was abolished by BoNT/C3 and Y-27632, a RhoA and Rho kinase inhibitor, respectively. The demyelination was equivalent between the preparations with and without dorsal root ganglion. LPA also caused a down-regulation of myelin proteins, such as myelin basic protein (MBP) and myelin protein zero (MPZ) to approximately 70% of control. All these findings suggest that the demyelination observed in the neuropathic pain due to nerve injury occurs through a direct action of LPA on Schwann cells.

Prothymosin- α plays a defensive role in retinal ischemia through necrosis and apoptosis inhibition

Prothymosin-α (ProTα) causes a switch in cell death mode from necrosis to neurotrophin-reversible apoptosis in primary cultured cortical neurons. In the present study, post-ischemic administration (3 or 24 h, intravenously) of recombinant mouse ProTα without neurotrophins completely prevented ischemia-induced retinal damage accompanying necrosis and apoptosis, as well as dysfunction assessed by electroretinogram. Treatments with anti-erythropoietin (EPO) or brain-derived neurotrophic factor (BDNF) immunoglobulin G (IgG) reversed ProTα-induced inhibition of apoptosis. ProTα upregulated retinal EPO and BDNF levels in the presence of ischemia. Moreover, intravitreous administration of anti-ProTα IgG or an antisense oligodeoxynucleotide for ProTα accelerated ischemia-induced retinal damage. We also observed that ischemia treatment caused a depletion of ProTα from retinal cells. Altogether, these results suggest that the systemic administration of ProTα switches ischemia-induced necrosis to apoptosis, which in turn is inhibited by neurotrophic factors upregulated by ProTα and ischemia. ProTα released upon ischemic stress was found to have a defensive role in retinal ischemia.

Lysophosphatidic acid‐induced membrane ruffling and brain‐derived neurotrophic factor gene expression are mediated by ATP release in primary microglia

We examined the effects of lysophosphatidic acid (LPA) on microglia, which may play an important role in the development and maintenance of neuropathic pain. LPA caused membrane ruffling as detected by scanning electron microscopy, and increased the expression of brain‐derived neurotrophic factor (BDNF) in a primary culture of rat microglia, which express LPA3, but not LPA1 or LPA2 receptors. These actions were inhibited by a Gαq/11‐antisense oligodeoxynucleotide (AS‐ODN), U73122, an inhibitor of phospholipase C (PLC), and apyrase, which specifically degrades ATP and ADP. When ATP release was measured using a luciferin‐luciferase bioluminescence assay, LPA was shown to increase it in an LPA3 and PLC inhibitor‐reversible manner. However, LPA‐induced ATP release was also blocked by the Gαq/11 AS‐ODN, but not by pertussis toxin. These results suggest that LPA induces the release of ATP from rat primary cultured microglia via the LPA3 receptor, Gαq/11 and PLC, and that the released ATP or ectopically converted ADP may in turn cause membrane ruffling via P2Y12 receptors and Gαi/o activation, and BDNF expression via activation of P2X4 receptors.

Identification of prothymosin-α1, the necrosis–apoptosis switch molecule in cortical neuronal cultures

We initially identified a nuclear protein, prothymosin-α1 (ProTα), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTα inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl2-family proteins. The apoptosis caused by ProTα was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTα-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCβII expression prevented this cell death mode switch. Collectively, these results suggest that ProTα is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.

Protein kinase C-mediated necrosis–apoptosis switch of cortical neurons by conditioned medium factors secreted under the serum-free stress

AbstractCortical neurons die in necrosis in the low-density (LD) culture, and in apoptosis in the high-density (HD) culture under the serum-free condition without any supplements. The neuronal death in LD culture was delayed by conditioned medium (CM) factors prepared from the HD culture. The CM switched the cell death mode from necrosis to apoptosis, characterized by various cell death markers and transmission electron microscopy. The CM inhibited the rapid decrease in cellular ATP levels and [3H]-2-deoxy glucose ([3H]-2-DG) uptake in the LD culture. Inhibitors of phospholipase C and protein kinase C effectively abolished the CM-induced elevation of survival activity, [3H]-2-DG uptake and ATP levels, and necrosis–apoptosis switch. All these results suggest that CM caused the cell death mode switch from necrosis to apoptosis through phospholipase C- and protein kinase C-mediated mechanisms.

Cell density-dependent death mode switch of cultured cortical neurons under serum-free starvation stress

Abstract1. Cell death mode switch of cortical neurons from E17 rats was studied. Cells rapidly died under the serum-free condition. The time-course of cell death was markedly delayed by increasing cell density for primary culture in the trypan blue exclusion, LDH release, and MTT assays.2. By analyzing cell death by the use of double staining using PI/TUNEL and PI/Annexin V combinations, the mode in the low density culture was found to be necrosis, while that in the high density culture was apoptosis.3. The intracellular ATP level after the start of serum-free culture rapidly decline to 25% of 0-time level in the low density culture, but it was 60% in the high density culture. Both oligomycin and zVAD-fmk markedly decreased ATP levels and the population of TUNEL-positive neurons, while 3-aminobenzamide slightly increased these indices.4. Thus, it is strongly suggested that the cell death mode switch from necrosis to apoptosis is closely related to intracellular ATP levels, and some conditioned medium factors observed in the high density culture may affect both ATP level and cell death mode switch.

Protein kinase C-mediated cell death mode switch induced by high glucose

AbsheadCortical neurons rapidly die in necrosis due to poor glucose uptake in the low-density (LD) culture under serum-free condition without any supplements. The scanning and transmission electron microscopical analyses characterized the necrosis by membrane disruption, mitochondrial swelling and loss of cytoplasmic electron density. High-glucose treatment delayed the neuronal death by suppressing necrosis, but induced apoptosis through increase in Bax levels, cytochrome c release, caspase-3 activation and DNA ladder formation. Although pyruvate as well as high glucose inhibited necrotic cell death and rapid decrease in cellular ATP levels, possibly related to decreased [3H]-2-deoxy glucose uptake under the serum-free condition, it did not induce apoptosis. Protein kinase C inhibitors blocked these changes related to the cell death mode switch. Several neurotrophic factors did not affect the necrosis, but potentiated high-glucose-induced survival activity, while inhibiting cytochrome c release. All these results suggest that high-glucose treatment causes neuronal cell death mode switch by inhibiting necrosis, while inducing apoptosis, which is prevented by neurotrophic factors.

Novel type of Gq/11 protein-coupled neurosteroid receptor sensitive to endocrine disrupting chemicals in mast cell line (RBL-2H3)

1 Agonistic neurosteroids, including pregnenolone, dehydroepiandrosterone and its sulfate (DHEAS), caused rapid degranulation in measurements of β‐hexosaminidase (β‐HEX) release from a mast cell line, RBL‐2H3. This degranulation was blocked by BSA‐conjugated progesterone (PROG‐BSA) or 17β‐estradiol, both of which are antagonistic neurosteroids. 2 DHEAS‐induced β‐HEX release was blocked by U‐73122 or xestospongin C, but not by PTX or EGTA. DHEAS‐induced β‐HEX release was also abolished by Gq/11‐AS, but not by Gq/11‐MS. Pharmacological analyses revealed that the neurosteroids stimulated a putative membrane receptor through activation of the novel Gq/11 and phospholipase C. 3 While representative endocrine‐disrupting chemicals (EDCs) did not show any degranulation or nocifensive actions by themselves, they blocked the DHEAS‐induced degranulation. 4 The binding of a PROG‐BSA‐fluorescein isothiocyanate conjugate (PROG‐BSA‐FITC) to cells was inhibited by neurosteroids and EDCs. 5 In the algogenic‐induced biting and licking responses test, DHEAS caused agonistic nocifensive actions in a dose‐dependent manner between 1 and 10 fmol (i.pl.). DHEAS‐induced nocifensive actions were abolished by PROG‐BSA or nonylphenol. 6 Taken together, these results suggest that a Gq/11‐coupled neurosteroid receptor may regulate the neuroimmunological activity related to sensory stimulation and that some EDCs have antagonistic actions for this receptor.

(-)1-(Benzofuran-2-yl)-2-propylaminopentane shows survival effect on cortical neurons under serum-free condition through sigma receptors.

AbstractSUMMARY 1. The rapid cell death of cortical neurons in serum-free culture was rescued by the condition medium from the high-density culture, but not by brain-derived neurotrophic factor or basic fibroblast growth factor.2. Similar rescue was observed by the addition of (−)BPAP, an impulse enhancer, and (+)-pentazocine, a sigma receptor agonist. These actions were blocked by BD1063, a sigma receptor antagonist.3. (−)BPAP showed a weak displacement activity in the [3H]pentazocine binding to synaptic membranes from rat cerebral cortex.4. These findings suggest that (−)BPAP and (+)-pentazocine have unique survival activity on cortical neurons through sigma receptors.