Expression of Amyloid Precursor Protein, Caveolin-1, Alpha-, Beta-, and Gamma-Secretases in Penumbra Cells after Photothrombotic Stroke and Evaluation of Neuroprotective Effect of Secretase and Caveolin-1 Inhibitors.

Our studies reveal changes in the expression of the main participants in the processing of amyloid precursor protein (APP) in neurons and astrocytes after photothrombotic stroke (PTS). Here we show the increase in the level of N- and C-terminal fragments of APP in the cytoplasm of ischemic penumbra cells at 24 h after PTS and their co-immunoprecipitation with caveolin-1. The ADAM10 α-secretase level decreased in the rat brain cortex on the first day after PTS. Levels of γ-secretase complex proteins presenilin-1 and nicastrin were increased in astrocytes, but not in neurons, in the penumbra after PTS. Inhibitory analysis showed that these changes lead to neuronal death and activation of astrocytes in the early recovery period after PTS. The caveolin-1 inhibitor daidzein shifted APP processing towards Aß synthesis, which caused astroglial activation. γ-secretase inhibitor DAPT down-regulated glial fibrillary acidic protein (GFAP) in astrocytes, prevented mouse cerebral cortex cells from PTS-induced apoptosis, and reduced the infarction volume. Thus, new generation γ-secretase inhibitors may be considered as potential agents for the treatment of stroke.

Histone Methyltransferases SUV39H1 and G9a and DNA Methyltransferase DNMT1 in Penumbra Neurons and Astrocytes after Photothrombotic Stroke.

BACKGROUND: Cerebral ischemia, a common cerebrovascular disease, is one of the great threats to human health and new targets for stroke therapy are needed. The transcriptional activity in the cell is regulated by epigenetic processes such as DNA methylation/demethylation, acetylation/deacetylation, histone methylation, etc. Changes in DNA methylation after ischemia can have both neuroprotective and neurotoxic effects depending on the degree of ischemia damage, the time elapsed after injury, and the site of methylation. METHODS: In this study, we investigated the changes in the expression and intracellular localization of DNA methyltransferase DNMT1, histone methyltransferases SUV39H1, and G9a in penumbra neurons and astrocytes at 4 and 24 h after stroke in the rat cerebral cortex using photothrombotic stroke (PTS) model. Methods of immunofluorescence microscopy analysis, apoptosis analysis, and immunoblotting were used. Additionally, we have studied the effect of DNMT1 and G9a inhibitors on the volume of PTS-induced infarction and apoptosis of penumbra cells in the cortex of mice after PTS. RESULTS: This study has shown that the level of DNMT1 increased in the nuclear and cytoplasmic fractions of the penumbra tissue at 24 h after PTS. Inhibition of DNMT1 by 5-aza-2'-deoxycytidine protected cells of PTS-induced penumbra from apoptosis. An increase in the level of SUV39H1 in the penumbra was found at 24 h after PTS and G9a was overexpressed at 4 and 24 h after PTS. G9a inhibitors A-366 and BIX01294 protected penumbra cells from apoptosis and reduced the volume of PTS-induced cerebral infarction. CONCLUSION: Thus, the data obtained show that DNA methyltransferase DNMT1 and histone methyltransferase G9a can be potential protein targets in ischemic penumbra cells, and their inhibitors are potential neuroprotective agents capable of protecting penumbra cells from postischemic damage to the cerebral cortex.

Histone Deacetylases and Their Isoform-Specific Inhibitors in Ischemic Stroke.

Cerebral ischemia is the second leading cause of death in the world and multimodal stroke therapy is needed. The ischemic stroke generally reduces the gene expression due to suppression of acetylation of histones H3 and H4. Histone deacetylases inhibitors have been shown to be effective in protecting the brain from ischemic damage. Histone deacetylases inhibitors induce neurogenesis and angiogenesis in damaged brain areas promoting functional recovery after cerebral ischemia. However, the role of different histone deacetylases isoforms in the survival and death of brain cells after stroke is still controversial. This review aims to analyze the data on the neuroprotective activity of nonspecific and selective histone deacetylase inhibitors in ischemic stroke.

The Role of Post-Translational Acetylation and Deacetylation of Signaling Proteins and Transcription Factors after Cerebral Ischemia: Facts and Hypotheses.

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) regulate transcription and the most important functions of cells by acetylating/deacetylating histones and non-histone proteins. These proteins are involved in cell survival and death, replication, DNA repair, the cell cycle, and cell responses to stress and aging. HDAC/HAT balance in cells affects gene expression and cell signaling. There are very few studies on the effects of stroke on non-histone protein acetylation/deacetylation in brain cells. HDAC inhibitors have been shown to be effective in protecting the brain from ischemic damage. However, the role of different HDAC isoforms in the survival and death of brain cells after stroke is still controversial. HAT/HDAC activity depends on the acetylation site and the acetylation/deacetylation of the main proteins (c-Myc, E2F1, p53, ERK1/2, Akt) considered in this review, that are involved in the regulation of cell fate decisions. Our review aims to analyze the possible role of the acetylation/deacetylation of transcription factors and signaling proteins involved in the regulation of survival and death in cerebral ischemia.

Localization and Expression of Sirtuins 1, 2, 6 and Plasticity-Related Proteins in the Recovery Period after a Photothrombotic Stroke in Mice.

Sirtuins, class III histone deacetylases, are involved in the regulation of tissue repair processes and brain functions after a stroke. The ability of some isoforms of sirtuins to circulate between the nucleus and cytoplasm may have various pathophysiological effects on the cells. In present work, we focused on the role of non-mitochondrial sirtuins SIRT1, SIRT2, and SIRT6 in the restoration of brain cells following ischemic stroke. Here, using a photothrombotic stroke (PTS) model in mice, we studied whether local stroke affects the level and intracellular localization of SIRT1, SIRT2, and SIRT6 in neurons and astrocytes of the intact cerebral cortex adjacent to the ischemic ipsilateral hemisphere and in the analogous region of the contralateral hemisphere at different time points during the recovery period after a stroke. We evaluated the co-localization of sirtuins with growth-associated protein-43 (GAP-43), the presynaptic marker synaptophysin (SYN) and acetylated α-tubulin (Ac-α-Tub), that are associated with brain plasticity and are known to be involved in brain repair after a stroke. The results show that during the recovery period, an increase in SIRT1 and SIRT2 levels occurred. The increase of SIRT1 level was associated with an increase in synaptic plasticity proteins, whereas the increase of SIRT2 level was associated with an acetylated of α-tubulin, that can reduce the mobility of neurites. SIRT6 co-localized with GAP-43, but not with SYN. Moreover, we showed that SIRT1, SIRT2, and SIRT6 are not involved in the PTS-induced apoptosis of penumbra cells. Taken together, our results suggest that sirtuins functions differ depending on cell type, intracellular localization, specificity of sirtuins isoforms to different substrates and nature of post-translational modifications of enzymes.

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.

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.

Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin.

Epigenetic processes play a critical role in melanoma development. However, little is known about proteins responsible for epigenetic transformations in melanoma cells. The processes in the peritumoral skin within the excision margin are almost unstudied. We studied the changes in expression of 112 proteins involved in epigenetic regulation of gene expression in the human cutaneous melanoma and its peritumoral zone using "The Proteomic Antibody Microarrays" (GRAA2, Sigma-Aldrich). Dimethylated histone H3 at lysines 4 and 9 as well as proteins involved in the regulation of transcription (histone deacetylases HDAC-1 and HDAC-11, DNA methyl-binding protein Kaiso), cell cycle control (protein kinases Aurora-В and PKR, chromosome protein CENP-E , and phosphorylated and acetylated histone H3), DNA repair (phosphorylated histone H2AX), and nuclear protein import (importins α3 and α5/7) were over-expressed in the melanoma tissue as compared with normal skin. At the same time, HDAC-10 and proliferating cell nuclear antigen PCNA were downregulated. In the peritumoral skin, at the excision margin (1-2 cm from the melanoma edge), we observed similar changes in expression of these proteins and, additionally, over-expression of arginine methyltransferases PRMT5 and NAD-dependent histone deacetylase SIR2. Histone methyltransferase G9a and metastasis-associated protein 2 were downregulated. Therefore, epigenetic regulation that requires histone modifications and expression of some regulatory proteins is of importance for melanoma development and propagation. The observed changes in the peritumoral skin may indicate the epigenetic pre-tuning in this zone possibly involved in malignant transformation. These results can be potentially useful for melanoma diagnostics and targeted therapy.