The Localization of p53 in the Crayfish Mechanoreceptor Neurons and Its Role in Axotomy-Induced Death of Satellite Glial Cells Remote from the Axon Transection Site.

Neuron and glia death after axon transection is regulated by various signaling proteins. Protein p53 is a key regulator of diverse cell functions including stress response, DNA repair, proliferation, and apoptosis. We showed that p53 was overexpressed in crayfish ganglia after bilateral axotomy. In the isolated crayfish stretch receptor, a simple natural neuroglial preparation, which consists of a single mechanoreceptor neuron (MRN) enveloped by glial cells, p53 regulated axotomy-induced death of glial cells remote from the axon transection site. In MRN, p53 immunofluorescence was highest in the nucleolus and in the narrow cytoplasmic ring around the nucleus; its levels in the nucleus and cytoplasm were lower. After axotomy, p53 accumulated in the neuronal perikaryon. Its immunofluorescence also increased in the neuronal and glial nuclei. However, p53 immunofluorescence in the most of neuronal nucleoli disappeared. Axotomy-induced apoptosis of remote glial cells increased in the presence of p53 activators WR-1065 and nutlin-3 but reduced by pifithrin-α that inhibits transcriptional activity of p53. Pifithrin-µ that inhibits p53 effect on mitochondria increased axotomy-induced apoptosis of remote glial cells but reduced their necrosis. Therefore, axotomy-induced apoptosis of remote glial cells was associated with p53 effect on transcription processes, whereas glial necrosis was rather associated with transcription-independent p53 effect on mitochondria. Apparently, the fate of remote glial cells in the axotomized crayfish stretch receptor is determined by the balance between different modalities of p53 activity.

Ca2+ mediates axotomy-induced necrosis and apoptosis of satellite glial cells remote from the transection site in the isolated crayfish mechanoreceptor.

Severe nerve injury such as axotomy induces neuron degeneration and death of surrounding glial cells. Using a crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glia, we showed that axotomy not only mechanically injures glial cells at the transection location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. We studied Ca2+role in spontaneous or axotomy-induced death of remote glial cells. Stretch receptors were isolated using the original technique that kept the neuron connected to the ventral cord ganglion (control preparations). Using Ca2+-sensitive fluorescence probe fluo-4, we showed Ca2+ accumulation in neuronal perikarion and glial envelope. Ca2+ gradually accumulated in glial cells after axotomy. In saline with triple Ca2+ concentration the axotomy-induced apoptosis of glial cells increased, but spontaneous or axotomy-induced necrosis was unexpectedly reduced. Saline with 1/3[Ca2+], oppositely, enhanced glial necrosis. Application of ionomycin, CdCl2, thapsigargin, and ryanodine showed the involvement of Ca2+ influx through ionic channels in the plasma membrane, inhibition of endoplasmic reticulum Ca2+-ATPase, and Ca2+ release from endoplasmic reticulum through ryanodine receptors in axotomy-induced glial necrosis. Apoptosis of glial cells surrounding axotomized neurons was promoted by ionomycin and thapsigargin. Possibly, other Ca2+ sources such as penetration through the plasma membrane contributed to axotomy-induced apoptosis and necrosis of remote glial cells. Thus, modulating different pathways that maintain calcium homeostasis, one can modulate axotomy-induced death of glial cells remote from the transection site.

On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells.

Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

Protection of the Crayfish Mechanoreceptor Neuron and Glial Cells from Photooxidative Injury by Modulators of Diverse Signal Transduction Pathways.

Oxidative stress is the reason of diverse neuropathological processes. Photodynamic therapy (PDT), an effective inducer of oxidative stress, is used for cancer treatment, including brain tumors. We studied the role of various signaling pathways in photodynamic injury and protection of single neurons and satellite glial cells in the isolated crayfish mechanoreceptor. It was photosensitized with alumophthalocyanine Photosens in the presence of inhibitors or activators of various signaling proteins. PDT eliminated neuronal activity and killed neurons and glial cells. Inhibitory analysis showed the involvement of protein kinases Akt, glycogen synthase kinase-3ß (GSK-3ß), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinases 1 and 2 (MEK1/2), calmodulin, calmodulin-dependent kinase II (CaMKII), adenylate cyclase, and nuclear factor NF-κB in PDT-induced necrosis of neurons. Nitric oxide (NO) and glial cell-derived neurotrophic factor (GDNF) reduced neuronal necrosis. In glial cells, protein kinases Akt, calmodulin, and CaMKII; protein kinases C and G, adenylate cyclase, and p38; and nuclear transcription factor NF-κB also mediated PDT-induced necrosis. In contrast, NO and neurotrophic factors nerve growth factor (NGF) and GDNF demonstrated anti-necrotic activity. Phospholipase Cγ, protein kinase C, GSK-3ß, mTOR, NF-κB, mitochondrial permeability transition pores, and NO synthase mediated PDT-induced apoptosis of glial cells, whereas protein kinase A, tyrosine phosphatases, and neurotrophic factors NGF, GDNF, and neurturin were involved in protecting glial cells from photoinduced apoptosis. Signaling pathways that control cell survival and death differed in neurons and glia. Inhibitors or activators of some signaling pathways may be used as potential protectors of neurons and glia from photooxidative stress and following death.

The method of isolation of the crayfish abdominal stretch receptor maintaining a connection of the sensory neuron to the ventral nerve cord ganglion.

The crayfish stretch receptor consisting of the single mechanoreceptor neurons enveloped by satellite glial cells is the simplest functioning neuroglial preparation. However, during isolation, its axons are usually transected that eliminates afferent regulation and induces complex axotomy-related signaling responses in neurons and satellite glia. We developed new microsurgical method of crayfish stretch receptor isolation, which preserves connections of sensory neurons to the ventral nerve cord ganglion. The stretch receptor may either remain on the abdominal carapace, or be completely isolated. In both cases, it may be either intact, or axotomized. The integrity of axons was confirmed by firing recording from proximal and distal axon points. Normal, necrotic and apoptotic cells were visualized using double fluorochroming with Hoechst 33342 and propidium iodide. The isolated mechanoreceptor neurons maintain regular firing during 8-10 or more hours. Glial cells surrounding non-axotomized neurons demonstrate lower necrosis and apoptosis levels than the axotomized ones. Unlike the existing method, in which the sensory neurons were axotomized, the present method preserves links between the sensory neurons and the ganglion and makes possible to avoid consequences of axotomy in neurons and satellite glia. The present neuroglial preparation may be used as a simple but informative model object in studies of axotomy-induced degeneration and survival of peripheral neurons, the role of glia in neuron injury, the signaling mechanisms of neuroglial interactions, and the effects of diverse physical and chemical factors on neuronal and glial cells.

Involvement of nitric oxide in photodynamic injury of neurons and glial cells.

Photodynamic therapy (PDT) is a potential tool for treatment of brain tumors. However, not only malignant but also healthy neurons and glial cells may be damaged during PDT. Nitric oxide is an important modulator of cell viability and intercellular neuroglial communications. In order to study its role in photodynamic injury of normal neurons and surrounding glial cells, we used the crayfish stretch receptor that consists of only two identified sensory neurons enveloped by glial cells. Photodynamic treatment with alumophthalocyanine Photosens and diode laser (670 nm, 0.4 W/cm(2)) induced firing elimination, necrosis of neurons and glia, and apoptosis of glial cells. NO generated by exogenous generators NONOate or sodium nitroprussside protected neurons and glial cells from PDT-induced necrosis but enhanced PDT-induced apoptosis of glial cells. Application of various inhibitors of NO synthase showed that the anti-necrotic effect of NO could be related, at least in glial cells, to its production by neuronal rather than inducible isoform of this enzyme. Unlike, the pro-apoptotic effect of NO on glial cells could be, at least in part, associated with inducible NO synthase. The proapoptotic effect of NO on glial cells could be mediated by protein kinase G, which is activated by NO-dependent production of cGMP, because it inhibition reduced the PDT-induced glial apoptosis.

On the role of phosphatidylinositol 3-kinase, protein kinase b/Akt, and glycogen synthase kinase-3ß in photodynamic injury of crayfish neurons and glial cells.

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3ß participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3ß pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3ß, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.