HDAC1 Expression, Histone Deacetylation, and Protective Role of Sodium Valproate in the Rat Dorsal Root Ganglia After Sciatic Nerve Transection.

Nerve injury is an important reason of human disability and death. We studied the role of histone deacetylation in the response of the dorsal root ganglion (DRG) cells to sciatic nerve transection. Sciatic nerve transection in the rat thigh induced overexpression of histone deacetylase 1 (HDAC1) in the ipsilateral DRG at 1-4 h after axotomy. In the DRG neurons, HDAC1 initially upregulated at 1 h but then redistributed from the nuclei to the cytoplasm at 4 h after axotomy. Histone H3 was deacetylated at 24 h after axotomy. Deacetylation of histone H4, accumulation of amyloid precursor protein, a nerve injury marker, and GAP-43, an axon regeneration marker, were observed in the axotomized DRG on day 7. Neuronal injury occurred on day 7 after axotomy along with apoptosis of DRG cells, which were mostly the satellite glial cells remote from the site of sciatic nerve transection. Administration of sodium valproate significantly reduced apoptosis not only in the injured ipsilateral DRG but also in the contralateral ganglion. It also reduced the deacetylation of histones H3 and H4, prevented axotomy-induced accumulation of amyloid precursor protein, which indicated nerve injury, and overexpressed GAP-43, a nerve regeneration marker, in the axotomized DRG. Therefore, HDAC1 was involved in the axotomy-induced injury of DRG neurons and glial cells. HDAC inhibitor sodium valproate demonstrated the neuroprotective activity in the axotomized DRG.

Са2+- and NF-κB-dependent generation of NO in the photosensitized neurons and satellite glial cells.

Photodynamic therapy (PDT) is used for killing of malignant cells in tumors including brain cancer. It can also damage normal neurons and glial cells. Nitric oxide (NO) is known to control PDT-induced cell death. To study the mechanisms that regulate NO generation in photosensitized neurons and glial cells, we used a simple model object - isolated crayfish mechanoreceptor that consists of a single sensory neuron surrounded by glial cells. PDT induced NO generation in glial cells, neuronal dendrites, and, less, in soma and axon. Using modulators of the cytosolic Ca2+ level and nuclear factor-kappa B (NF-κB) activity, we showed that Ca2+ and NF-κB regulate NO generation in the photosensitized neurons and glia. Actually, NO production was stimulated by 4-fold cadmium chloride (CdCl2) concentration in the saline, Ca2+ ionophore ionomycine, or inhibition of Ca2+-ATPase in the endoplasmic reticulum by 2,5-ditert-butylbenzene-1,4-diol (tBuBHQ). Oppositely, CdCl2 or nifedipine, blockers of Ca2+ channels in the plasma membrane, decreased NO generation. NO production was also inhibited by S-methylthiouronium sulfate (SMT), inhibitor of Ca2+-independent inducible NO synthase. SMT also prevented the stimulation of PDT-induced NO generation by NF-κB activator prostratin. This suggests the involvement of both Ca2+-dependent neuronal NO synthase and Ca2+-independent inducible NO synthase, which is regulated by NF-κB, in NO production in the crayfish neurons and glia.

Apoptosis regulation in the penumbra after ischemic stroke: expression of pro- and antiapoptotic proteins.

Ischemic stroke is the leading cause of human disability and mortality in the world. The main problem in stroke therapy is the search of efficient neuroprotector capable to rescue neurons in the potentially salvageable transition zone (penumbra), which is expanding after brain damage. The data on molecular mechanisms of penumbra formation and expression of diverse signaling proteins in the penumbra during first 24 h after ischemic stroke are discussed. Two basic features of cell death regulation in the ischemic penumbra were observed: (1) both apoptotic and anti-apoptotic proteins are simultaneously over-expressed in the penumbra, so that the fate of individual cells is determined by the balance between these opposite tendencies. (2) Similtaneous and concerted up-regulation in the ischemic penumbra of proteins that execute apoptosis (caspases 3, 6, 7; Bcl-10, SMAC/DIABLO, AIF, PSR), signaling proteins that regulate different apoptosis pathways (p38, JNK, DYRK1A, neurotrophin receptor p75); transcription factors that control expression of various apoptosis regulation proteins (E2F1, p53, c-Myc, GADD153); and proteins, which are normally involved in diverse cellular functions, but stimulate apoptosis in specific situations (NMDAR2a, Par4, GAD65/67, caspase 11). Hence, diverse apoptosis initiation and regulation pathways are induced simultaneously in penumbra from very different initial positions. Similarly, various anti-apoptotic proteins (Bcl-x, p21/WAF-1, MDM2, p63, PKBα, ERK1, RAF1, ERK5, MAKAPK2, protein phosphatases 1α and MKP-1, estrogen and EGF receptors, calmodulin, CaMKII, CaMKIV) are upregulated. These data provide an integral view of neurodegeneration and neuroprotection in penumbra. Some discussed proteins may serve as potential targets for anti-stroke therapy.

The Focal-Focal Preconditioning Effect of Photothrombotic Impact on the Signaling Protein Profile in the Penumbra Surrounding the Ischemic Core Induced by Another Photothrombotic Impact.

Ischemic tolerance is the establishment of brain resistance to severe ischemic damage by a mild preconditioning stimulus, insufficient to irreversible tissue damage, but capable of initiating a defense response. We developed the model of focal-focal ischemic tolerance, in which the first local photothrombotic infarct (PTI) in the rat brain cortex reduced the infarct caused by second PTI applied to the contralateral cortex of the same rat 7 days later. Using antibody microarrays, we compared protein profiles in the penumbra surrounding the PTI core after single and double PTI. We observed up- or downregulation of several dozens of proteins that are aimed at neurodegeneration or neuroprotection. Both single and double PTI induced damaging processes in the rat cerebral cortex that included over-expression of various pro-apoptotic and signaling proteins and downregulation of other signaling proteins and regulators of proliferation, some components of actin, intermediate fiber and microtubular cytoskeletons, and proteins involved in vesicle transport and synaptic transmission. The simultaneous protective processes included the upregulation of different signaling and anti-apoptotic proteins, stimulators of proliferation, and proteins involved in remodeling of actin cytoskeleton. The elevated expression of some signaling proteins, such as calcium-dependent PLCγ1, PKVα1, CaMKIIα, calnexin, and calreticulin was preserved after double PTI. Less pro-survival proteins were downregulated in the penumbra after double than single impact.

Photo-Induced Oxidative Stress Impairs Mitochondrial Metabolism in Neurons and Astrocytes.

Photodynamic therapy is selective destruction of cells stained with a photosensitizer upon irradiation with light at a specific wavelength in the presence of oxygen. Cell death upon photodynamic treatment is known to occur mainly due to free radical production and subsequent development of oxidative stress. During photodynamic therapy of brain tumors, healthy cells are also damaged; considering this, it is important to investigate the effect of the treatment on normal neurons and glia. We employed live-cell imaging technique to investigate the cellular mechanism of photodynamic action of radachlorin (200 nM) on neurons and astrocytes in primary rat cell culture. We found that the photodynamic effect of radachlorin increases production of reactive oxygen species measured by dihydroethidium and significantly decrease mitochondrial membrane potential. Mitochondrial depolarization was independent of opening of mitochondrial permeability transition pore and was insensitive to blocker of this pore cyclosporine A. However, irradiation of cells with radachlorin dramatically decreased NADH autofluorescence and also reduced mitochondrial NADH pool suggesting inhibition of mitochondrial respiration by limitation of substrate. This effect could be prevented by inhibition of poly (ADP-ribose) polymerase (PARP) with DPQ. Thus, irradiation of neurons and astrocytes in the presence of radachlorin leads to activation of PARP and decrease in NADH that leads to mitochondrial dysfunction.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).

Photothrombotic Stroke as a Model of Ischemic Stroke.

The search of effective anti-stroke neuroprotectors requires various stroke models adequate for different aspects of the ischemic processes. The photothrombotic stroke model is particularly suitable for the study of cellular and molecular mechanisms underlying neurodegeneration, neuroprotection, and neuroregeneration. It is a model of occlusion of small cerebral vessels, which provides detailed study of molecular mechanisms of ischemic cell death and useful for search of potential anti-stroke agents. Its advantages include well-defined location and size of ischemic lesion that are determined by the aiming of the laser beam at the predetermined brain region; easy impact dosing by changing light intensity and duration; low invasiveness and minimal surgical intervention without craniotomy and mechanical manipulations with blood vessel, which carry the risk of brain trauma; low animal mortality and prolonged sensorimotor impairment that provide long-term study of stroke consequences including behavior impairment and recovery; independence on genetic variations of blood pressure and vascular architecture; and high reproducibility. This review describes the current application of the photothrombotic stroke model for the study of cellular and molecular mechanisms of stroke development and ischemic penumbra formation, as well as for the search of anti-stroke drugs.

Photodynamic therapy-induced nitric oxide production in neuronal and glial cells.

Nitric oxide (NO) has been recently demonstrated to enhance apoptosis of glial cells induced by photodynamic therapy (PDT), but to protect glial cells from PDT-induced necrosis in the crayfish stretch receptor, a simple neuroglial preparation that consists of a single mechanosensory neuron enveloped by satellite glial cells. We used the NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate to study the distribution and dynamics of PDT-induced NO production in the mechanosensory neuron and surrounding glial cells. The NO production in the glial envelope was higher than in the neuronal soma axon and dendrites both in control and in experimental conditions. In dark NO generator, DEA NONOate or NO synthase substrate L-arginine hydrochloride significantly increased the NO level in glial cells, whereas NO scavenger 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) or inhibitors of NO synthase L-NG-nitro arginine methyl ester and N?-nitro-L-arginine decreased it. PDT induced the transient increase in NO production with a maximum at 4 to 7 min after the irradiation start followed by its inhibition at 10 to 40 min. We suggested that PDT stimulated neuronal rather than inducible NO synthase isoform in glial cells, and the produced NO could mediate PDT-induced apoptosis.

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.

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.

Signal transduction in human cutaneous melanoma and target drugs.

Malignant melanoma is an extremely aggressive and metastatic cancer, highly resistant to conventional treatment modalities. Understanding of fundamental mechanisms responsible for its genesis and progression is critical for development of successful chemotherapeutic treatment. It is becoming clear that melanoma results from complex changes in multiple signaling pathways that control cell proliferation and ability to evade the cell death processes. Impairment or hyper-activation of some components of these pathways may lead to malignant transformation and cancer development. In the present review we consider the current data on involvement of such signaling pathways as cyclin/CDK, Ras/Raf/MEK/MAPK, JNK/c-Jun/AP-1, PI3K/Akt/PTEN/mTOR, IKK/I-κB/NF-κB, Wnt/ß-catenin, Notch, Jak/STAT, MITF and some growth factors in regulation of the cell cycle progression, apoptosis and development of human cutaneous melanoma. Understanding of molecular aberrations that underlie melanoma oncogenesis is essential for improvement of diagnosis, accurate prognosis assessment, and rational design of effective therapeutics. Inhibitors of these pathways may serve as promising tools for anti-melanoma targeted therapy. Some novel anti-melanoma target drugs are characterized.

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.

Elevated activity of the crayfish stretch receptor neuron increases resistance of surrounding glial cells to apoptosis induced by photodynamic treatment.

Neuroglial interaction is very important for functioning and survival of nerve and glial cells. In the present work, we studied the influence of the intense neuronal activity on survival of the isolated crayfish stretch receptor neuron and surrounding glial cells subjected to photodynamic treatment, which induces intense oxidative stress. In the experimental group, neurons were stimulated by multiple extensions of the receptor muscle for 1h so that the firing rate did not fall below 10-15 Hz, whereas in the control group, the receptor muscles were relaxed and neurons were silent. After stimulation, the preparations were photosensitized with alumophthalocyanine Photosens and irradiated by 670 nm laser diode. The isolated stretch receptors were stained with propidium iodide and Hoechst 33342, which reveal the nuclei of the necrotic and the apoptotic cells, respectively. The level of apoptosis of photosensitized glial cells was significantly lower in the experimental group compared to the resting control. Necrosis of neurons and glial cells was not significantly influenced. Therefore, elevated neuronal activity increased the resistance of the surrounding glial cells to photoinduced apoptosis. This could be attributed to the depletion of the energetic resources, which are transferred from glia into the neuron to support its firing, or to the neurotrophic neuron-to-glia signaling.

Ultrastructure of neuroglial contacts in crayfish stretch receptor.

In order to explore neuroglial relationships in a simple nervous system, we have studied the ultrastructure of the crayfish stretch receptor, which consists of only two mechanoreceptor neurons enwrapped by glial cells. The glial envelope comprises 10-30 glial layers separated by collagen sheets. The intercellular space between the neuronal and glial membranes is generally less than 10-15 nm in width. This facilitates diffusion between neurons and glia but restricts neuron communication with the environment. Microtubule bundles passing from the dendrites to the axon through the neuron body limit vesicular transport between the perikaryon and the neuronal membrane. Numerous invaginations into the neuron cytoplasm strengthen glia binding to the neuron and shorten the diffusion pathway between them. Double-membrane vesicles containing fragments of glial, but not neuronal cytoplasm, represent the captured tips of invaginations. Specific triads, viz., "flat submembrane cisterns - vesicles - mitochondria", are presumably involved in the formation of the invaginations and double-membrane vesicles and in neuroglial exchange. The tubular lattice in the glial cytoplasm might transfer ions and metabolites between the glial layers. The integrity of the neuronal and glial membranes is impaired in some places. However, free neuroglial passage might be prevented or limited by the dense diffuse material accumulated in these regions. Thus, neuroglial exchange with cellular components might be mediated by transmembrane diffusion, especially in the invaginations and submembrane cisterns, by the formation of double-walled vesicles in which large glial masses are captured and by transfer through tubular lattices.

Photodynamic effect of hypericin and a water-soluble derivative on isolated crayfish neuron and surrounding glial cells.

Hypericin (Hyp) has been proposed as a fluorochrome for fluorescence diagnostics and as a photosensitizer for photodynamic therapy of cancer. However, its insolubility in water is a serious drawback. A novel water-soluble hypericin derivative (Hyp-S) has been constructed, using polyvinylpyrrolidone as a carrier. We used the crayfish stretch receptor, consisting of receptor neuron and satellite glial cells, for comparison of the photodynamic effects of Hyp and Hyp-S. Hyp-S was more toxic in the dark than Hyp and inactivated the neurons at concentrations exceeding 4 microM while Hyp was toxic to the neurons only at the concentrations larger than 20 microM. Electrophysiological investigations revealed polyphasic neuron responses to photosensitization with Hyp as well as with Hyp-S (1 microM concentration, 30 min incubation; irradiation with filtered light from a lamp with an emission maximum near 600 nm and an intensity of 0.2 W/cm2). In the concentration range 1-4 microM Hyp-S was more phototoxic than Hyp. Fluorescence microscopy showed that both sensitizers were predominately localized in the glial envelope surrounding the neuron. A minor fraction of hypericin was found in the neuron perinuclear area rich in cytoplasm organelles. This suggests the potential application of Hyp and Hyp-S for visualization and selective photodynamic treatment of malignant gliomas.

Photodynamic inactivation of isolated crayfish neuron requires protein kinase C, PI 3-kinase and Ca2+.

Involvement of some signalling pathways in response to photodynamic therapy (PDT) of sulfonated aluminium phthalocyanine Photosens has been studied in isolated nerve cell. Neurone photosensitisation with 10(-7) M Photosens gradually inhibited firing and irreversibly abolished neuronal activity. Activation of protein kinase C (PKC) by phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) precipitated PDT-induced abolition of neurone activity and caused nucleus swelling and impairment of the nucleus border. Elevation of cytosolic Ca(2+) concentration by ionomycin or thapsigargin also reduced neurone lifetime. In contrast, the PKC inhibitors staurosporine, hypericin or chelerythrine as well as the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 increased neurone lifetime. These results showed that PKC, PI 3-kinase and Ca(2+) are involved in PDT-induced neurone inactivation and following death.