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.

NO-Dependent Mechanisms of p53 Expression and Cell Death in Rat's Dorsal Root Ganglia after Sciatic-Nerve Transection.

Peripheral-nerve injury is a frequent cause of disability. Presently, no clinically effective neuroprotectors have been found. We have studied the NO-dependent expression of p53 in the neurons and glial cells of the dorsal root ganglia (DRG) of a rat's spinal cord, as well as the role of NO in the death of these cells under the conditions of axonal stress, using sciatic-nerve axotomy as a model. It was found out that axotomy led to the nuclear-cytoplasmic redistribution of p53 in neurons, 24 h after trauma. The NO donor led to a considerable increase in the level of p53 in nuclei and, to a smaller degree, in the cytoplasm of neurons and karyoplasm of glial cells 4 and 24 h after axotomy. Application of a selective inhibitor of inducible NO-synthase (iNOS) provided the opposite effect. Introduction of the NO donor resulted in a significant increase in cell death in the injured ipsilateral DRG, 24 h and 7 days after trauma. The selective inhibitor of iNOS demonstrated a neuroprotective effect. Axotomy was shown to upregulate the iNOS in nuclei and cytoplasm of DRG cells. The NO-dependent expression of p53, which is particularly achieved through iNOS activation, is believed to be a putative signaling mechanism of neural and glial-cell death after axotomy.

Sirtuins 1 and 2 in the Acute Period After Photothrombotic Stroke: Expression, Localization and Involvement in Apoptosis.

Sirtuins (SIRTs) are NAD+- dependent histone deacetylases. They are involved in a variety of biological pathways and are thought to be a promising target for treating several human disorders. Although evidence is piling up to support the neuroprotective role of SIRTs in ischemic stroke, the role of different sirtuin isoforms needs further investigation. We studied the effects of photothrombotic stroke (PTS) on the expression and localization of sirtuins SIRT1 and SIRT2 in neurons and astrocytes of the penumbra and tested the activity of their selective and non-selective inhibitors. SIRT1 levels significantly decreased in the penumbra cells nuclei and increased in their cytoplasm. This indicated a redistribution of SIRT1 from the nucleus to the cytoplasm after PTS. The expression and intracellular distribution of SIRT1 were also observed in astrocytes. Photothrombotic stroke caused a sharp increase in SIRT2 levels in the cytoplasmic fraction of the penumbra neurons. SIRT2 was not expressed in the penumbra astrocytes. SIRT1 and SIRT2 did not co-localize with TUNEL-positive apoptotic cells. Mice were injected with EX-527, a selective SIRT1 inhibitor; SirReal2, selective SIRT2 inhibitor or salermide, a nonspecific inhibitor of SIRT1 and SIRT2. These inhibitors did not demonstrate any change in the infarction volume or the apoptotic index, compared to the control samples. The studies presented indicate the involvement of these sirtuins in the response of brain cells to ischemia in the first 24 h, but the alterations in their expression and change in the localization of SIRT1 are not related to the regulation of penumbra cell apoptosis in the acute period after PTS.

Histone Deacetylases as Epigenetic Targets for Treating Parkinson's Disease.

Parkinson's disease (PD) is a chronic progressive neurodegenerative disease that is increasingly becoming a global threat to the health and life of the elderly worldwide. Although there are some drugs clinically available for treating PD, these treatments can only alleviate the symptoms of PD patients but cannot completely cure the disease. Therefore, exploring other potential mechanisms to develop more effective treatments that can modify the course of PD is still highly desirable. Over the last two decades, histone deacetylases, as an important group of epigenetic targets, have attracted much attention in drug discovery. This review focused on the current knowledge about histone deacetylases involved in PD pathophysiology and their inhibitors used in PD studies. Further perspectives related to small molecules that can inhibit or degrade histone deacetylases to treat PD were also discussed.

E2F1 Expression and Apoptosis Initiation in Crayfish and Rat Peripheral Neurons and Glial Cells after Axonal Injury.

Neurotrauma is among the main causes of human disability and mortality. The transcription factor E2F1 is one of the key proteins that determine the fate of cells. The involvement of E2F1 in the regulation of survival and death of peripheral nerve cells after axotomy has not been previously studied. We, for the first time, studied axotomy-induced changes in the expression and localization of E2F1 following axonal injury in rats and crayfish. Immunoblotting and immunofluorescence microscopy were used for the analysis of the expression and intracellular localization of E2F1 and its changes after axotomy. To evaluate whether this transcription factor promotes cell apoptosis, we examined the effect of pharmacological inhibition of E2F activity in axotomized rat models. In this work, axotomy caused increased expression of E2F1 as early as 4 h and even 1 h after axotomy of mechanoreceptor neurons and ganglia of crayfish ventral nerve cord (VNC), as well as rat dorsal root ganglia (DRG). The level of E2F1 expression increased both in the cytoplasm and the nuclei of neurons. Pharmacological inhibition of E2F demonstrated a pronounced neuroprotective activity against axotomized DRGs. E2F1 and downstream targets could be considered promising molecular targets for the development of potential neuroprotective agents.

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 p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy.

The search for effective neuroprotective agents for the treatment of neurotrauma has always been of great interest to researchers around the world. Extracellular heat shock protein 70 (eHsp70) is considered a promising agent to study, as it has been demonstrated to exert a significant neuroprotective activity against various neurodegenerative diseases. We showed that eHsp70 can penetrate neurons and glial cells when added to the incubation medium, and can accumulate in the nuclei of neurons and satellite glial cells after axotomy. eHsp70 reduces apoptosis and necrosis of the glial cells, but not the neurons. At the same time, co-localization of eHsp70 with p53 protein, one of the key regulators of apoptosis, was noted. eHsp70 reduces the level of the p53 protein apoptosis promoter both in glial cells and in the nuclei and cytoplasm of neurons, which indicates its neuroprotective effect. The ability of eHsp70 to reverse the proapoptotic effect of the p53 activator WR1065 may indicate its ability to regulate p53 activity or its proteosome-dependent degradation.

The Expression of E2F1, p53, and Caspase 3 in the Rat Dorsal Root Ganglia After Sciatic Nerve Transection.

Neurotrauma is among the main causes of human disability and mortality. Nerve injury impairs not only neurons but also causes death of satellite glial cells remote from the injury site. We studied the dynamics of expression of different proapoptotic proteins (E2F1, p53, caspase 3) in the dorsal root ganglia (DRG) of a rat after sciatic nerve transection. TUNEL staining and immunoblotting were used for analysis of cell apoptosis and axotomy-induced biochemical changes. Apoptosis of glial cells was observed at 24 h after sciatic nerve transection and increased on day 7, when apoptosis of some neurons only started. The earliest proapoptotic event in the injured DRG was overexpression of transcription factor E2F1 at 4 h after sciatic nerve transection. This preceded the induction of p53 and cleavage of caspase 3 at 24-h post-axotomy. The nerve injury marker amyloid precursor protein and the nerve regeneration marker GAP-43 were overexpressed in DRG on day 7 after sciatic nerve transection. We also developed a novel fluorescence method for differential visualization of the rat DRG and nerves by means of double staining with propidium iodide and Hoechst 33342 that impart red and blue-green fluorescence, respectively. The present experiments showed that glial cells remote from the nerve transection site were more vulnerable to axotomy than DRG neurons. E2F1 and p53 may be considered promising molecular targets for development of potential neuroprotective agents.

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.

Сlass II histone deacetylases in the post-stroke recovery period-expression, cellular, and subcellular localization-promising targets for neuroprotection.

Histone deacetylases (HDAC) inhibitors can protect nerve cells after a stroke, but it is unclear which HDAC isoform is involved in this effect. We studied cellular and intracellular rearrangement of class II HDACs at late periods after photothrombotic infarct (PTI) in the mouse sensorimotor cortex in the tissue surrounding the ischemia core and in the corresponding region of the contralateral hemisphere. We observed a decrease in HDAC4 in cortical neurons and an increase in its nuclear translocation. HDAC6 expression in neurons was also increased. Moreover, HDAC6-positive cells had elevated apoptosis. Tubostatin A (Tub A)-induced decrease in the activity of HDAC6 restored acetylation of α-tubulin during the early poststroke recovery period and reduced apoptosis of nerve cells thus protecting the brain tissue. Selective inhibition of HDAC6 elevated expression of growth-associated protein-43 (GAP43), which remained high up to 14 days after stroke and promoted axogenesis and recovery from the PTI-induced neurological deficit. Selective HDAC6 inhibitor Tub A markedly reduced neuronal death and increased acetylation of α-tubulin and the level of GAP43. Thus, HDAC6 inhibition could be a promising strategy for modulation of brain recovery as it can increase the intensity and reduce the duration of reparation processes in the brain after stroke.

Axotomy-Induced Changes of the Protein Profile in the Crayfish Ventral Cord Ganglia.

We suggest novel experimental model of nerve injury-bilaterally axotomized ganglia of the crayfish ventral nerve cord (VNC). Using proteomic antibody microarrays, we showed upregulation of apoptosis execution proteins (Bcl-10, caspases 3, 6, and 7, SMAC/DIABLO, AIF), proapoptotic signaling proteins and transcription factors (c-Myc, p38, E2F1, p53, GADD153), and multifunctional proteins capable of initiating apoptosis in specific situations (p75, NMDAR2a) in the axotomized VNC ganglia. Simultaneously, anti-apoptotic proteins (p21WAF-1, MDM2, Bcl-x, Mcl-1, MKP1, MAKAPK2, ERK5, APP, calmodulin, estrogen receptor) were overexpressed. Some proteins associated with actin cytoskeleton (α-catenin, catenin p120CTN, cofilin, p35, myosin Vα) were upregulated, whereas other actin-associated proteins (ezrin, distrophin, tropomyosin, spectrin (α + ß), phosphorylated Pyk2) were downregulated. Various cytokeratins and ßIV-tubulin, components of intermediate filament and microtubule cytoskeletons, were also downregulated that could be the result of tissue destruction. Downregulation of proteins involved in clathrin vesicle formation (AP2α and AP2γ, adaptin (ß1 + ß2), and syntaxin) indicated impairment of vesicular transport and synaptic processes. The levels of L-DOPA decarboxylase, tyrosine, and tryptophan hydroxylases that mediate synthesis of serotonin, dopamine, norepinephrine, and epinephrine decreased. Overexpression of histone deacetylases HDAC1, HDAC2, and HDAC4 contributed to suppression of transcription and protein synthesis. So, the balance of multidirectional processes aimed either at cell death, or to repair and recovery, determines the cell fate. Present data provide integral, albeit incomplete, view on the nervous tissue response to axotomy. Some of these proteins can be probably potential markers of nerve injury and targets for neuroprotective therapy.