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Kloukina-Pantazidou I.,Neurobiology Research Institute | Havaki S.,Neurobiology Research Institute | Chrysanthou-Piterou M.,Neurobiology Research Institute | Chrysanthou-Piterou M.,National and Kapodistrian University of Athens | And 4 more authors.
Ultrastructural Pathology | Year: 2010

Studies of peripheral blood leukocytes of schizophrenic patients have shown in electron microscopy (EM) that decondensation of the chromatin constitutes a biological marker indicating increased genomic expression. Since this increase depends on chromatin relaxation by dissociation of lysine-rich histone H1 from nucleosomes, with exposure of arginine residues of core histones, the ratio of arginine to lysine residues in each nucleus represents a reliable measure of activation. Lysine- and arginine-rich proteins are demonstrable in light microscopy (LM), differentially, as yellow and black, respectively, with the ammoniacal silver reaction (ASR). Application of ASR on leukocyte pellets before they are dehydrated and embedded in epoxy resins gives reliable results in semithin sections. In thin sections the ASR method localizes only the amino acid arginine by forming deposits of electron-opaque particles, visualized in the EM. Leukocytes of 12 first-episode schizophrenic patients and 5 controls were used. Light micrographs of the semithin sections were inserted in a personal computer. The percentage of lysine and arginine was measured in 300 nuclei per subject. Morphometry showed that lymphocytes of schizophrenic patients have increased ratios of arginine to lysine, compared to controls, indicating activation; neutrophils of the patients have even a higher ratio, indicating an abnormal condition of the genome. Chromatin conformational changes are also evident by phosphotungstic acid hematoxylin (PTAH) block staining, which reveals condensed chromatin as an electron-lucent area in the nuclei, and decondensed chromatin as an electron-dense area. Because decondensed chromatin is a biological marker of schizophrenia, the efficacy of these methods to demonstrate this particular state offers a tool for early diagnosis, since first-episode schizophrenic patients have a better prognosis when treatment is started promptly, at the beginning of the disease. © 2010 Informa UK Ltd.


Kloukina-Pantazidou I.,Neurobiology Research Institute | Chrysanthou-Piterou M.,Neurobiology Research Institute | Chrysanthou-Piterou M.,National and Kapodistrian University of Athens | Havaki S.,Neurobiology Research Institute | And 3 more authors.
Ultrastructural Pathology | Year: 2013

Our previous histochemical and ultrastructural studies have identified, in human catecholamine neurons, abundant spherical acidophilic protein bodies (pb), which originate from regular mitochondria, retaining their double membrane. In locus coeruleus (LC) neurons, pb have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline, as demonstrated by immunolocalization of Dopamine-β-Hydroxylase. In the present study, in order to reinforce the identity of pb as monoamine storage sites in human LC, and to assess their potential of somatodendritic release, we studied the subcellular immunolocalization of chromogranin A (CgA) and vesicular monoamine transporter 2 (VMAT2), given the fact that their localization defines the vesicles capacity of filling with monoamine and hence exocytotic release. The data provided in the present study, demonstrate the novel ultrastructural immunolocalization of both CgA and VMAT2 in protein bodies, supporting their involvement in somatodendritic storage and release of noradrenaline in human LC. Since the molecular mechanism of LC somatodendritic exocytosis remains largely elusive, the present study may shed light to a better understanding of this mechanism. © Informa Healthcare USA, Inc.


Xilouri M.,Surgery Academy | Brekk O.R.,Surgery Academy | Brekk O.R.,University of Crete | Polissidis A.,Surgery Academy | And 5 more authors.
Autophagy | Year: 2016

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis. © 2016 Taylor & Francis.


Emmanouilidou E.,Academy of Athens | Minakaki G.,Friedrich - Alexander - University, Erlangen - Nuremberg | Keramioti M.V.,Academy of Athens | Xylaki M.,Academy of Athens | And 5 more authors.
Brain | Year: 2016

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of 7alpha;-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.


Piterou M.C.,National and Kapodistrian University of Athens | Pantazidou I.K.,Neurobiology Research Institute | Havaki S.,Neurobiology Research Institute | Havaki S.,National and Kapodistrian University of Athens | And 3 more authors.
In Vivo | Year: 2014

Background/Aim: The aim of this study was to investigate the state of chromatin condensation in peripheral blood leukocytes of alcoholics, during the early detoxification period, in order to highlight structural modifications, indicating epigenetic mechanisms regulated by alcohol. Materials and Methods: Blood samples were obtained from alcoholic patients, who were admitted for detoxification on an inpatient basis, and from healthy controls. The level of condensed heterochromatin and decondensed euchromatin were detected through the ratio of lysine to arginine residues, by the application of the ammoniacal silver reaction (ASR) staining on leukocyte pellets, and through immunohistochemical localization of histone H1 on peripheral blood smears. Results: Lymphocytes and neutrophils with relaxed de-condensed chromatin were found, indicating a more reactive genome in alcoholics, even at the stage of detoxification. Conclusion: The results underline the importance of chromatin structure of leukocytes as a sensitive, peripheral, biological marker for epigenetic studies in living chronic alcoholics.


PubMed | National and Kapodistrian University of Athens and Neurobiology Research Institute
Type: Journal Article | Journal: In vivo (Athens, Greece) | Year: 2014

The aim of this study was to investigate the state of chromatin condensation in peripheral blood leukocytes of alcoholics, during the early detoxification period, in order to highlight structural modifications, indicating epigenetic mechanisms regulated by alcohol.Blood samples were obtained from alcoholic patients, who were admitted for detoxification on an inpatient basis, and from healthy controls. The level of condensed heterochromatin and de-condensed euchromatin were detected through the ratio of lysine to arginine residues, by the application of the ammoniacal silver reaction (ASR) staining on leukocyte pellets, and through immunohistochemical localization of histone H1 on peripheral blood smears.Lymphocytes and neutrophils with relaxed de-condensed chromatin were found, indicating a more reactive genome in alcoholics, even at the stage of detoxification.The results underline the importance of chromatin structure of leukocytes as a sensitive, peripheral, biological marker for epigenetic studies in living chronic alcoholics.

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