Zivin M


Full name : Zivin Marko

First name : Marko

Mail : Laboratory for Brain Research, Institute of Pathophysiology, Medical Faculty University of Ljubljana, Ljubljana

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Country : Slovenia

Email : marko.zivin@mf.uni-lj.si

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References (21)

Title : Rat Group IIA Secreted Phospholipase A(2) Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease - Ivanusec_2022_Int.J.Mol.Sci_23_
Author(s) : Ivanusec A , Sribar J , Leonardi A , Zorovic M , Zivin M , Krizaj I
Ref : Int J Mol Sci , 23 : , 2022
Abstract : Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A(2) group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, beta-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom beta-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.
ESTHER : Ivanusec_2022_Int.J.Mol.Sci_23_
PubMedSearch : Ivanusec_2022_Int.J.Mol.Sci_23_
PubMedID: 36293221

Title : Pseudo-irreversible butyrylcholinesterase inhibitors: Structure-activity relationships, computational and crystallographic study of the N-dialkyl O-arylcarbamate warhead - Meden_2022_Eur.J.Med.Chem_247_115048
Author(s) : Meden A , Knez D , Brazzolotto X , Modeste F , Perdih A , Pislar A , Zorman M , Zorovic M , Denic M , Pajk S , Zivin M , Nachon F , Gobec S
Ref : Eur Journal of Medicinal Chemistry , 247 :115048 , 2022
Abstract : Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure-activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC(50) time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.
ESTHER : Meden_2022_Eur.J.Med.Chem_247_115048
PubMedSearch : Meden_2022_Eur.J.Med.Chem_247_115048
PubMedID: 36586299
Gene_locus related to this paper: human-BCHE

Title : Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes - Pajk_2020_J.Enzyme.Inhib.Med.Chem_35_498
Author(s) : Pajk S , Knez D , Kosak U , Zorovic M , Brazzolotto X , Coquelle N , Nachon F , Colletier JP , Zivin M , Stojan J , Gobec S
Ref : J Enzyme Inhib Med Chem , 35 :498 , 2020
Abstract : Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.
ESTHER : Pajk_2020_J.Enzyme.Inhib.Med.Chem_35_498
PubMedSearch : Pajk_2020_J.Enzyme.Inhib.Med.Chem_35_498
PubMedID: 31914836
Gene_locus related to this paper: human-BCHE

Title : The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity - Kosak_2018_J.Med.Chem_61_119
Author(s) : Kosak U , Brus B , Knez D , Zakelj S , Trontelj J , Pislar A , Sink R , Jukic M , Zivin M , Podkowa A , Nachon F , Brazzolotto X , Stojan J , Kos J , Coquelle N , Salat K , Colletier JP , Gobec S
Ref : Journal of Medicinal Chemistry , 61 :119 , 2018
Abstract : The enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimer's disease, thus classifying BChE as a promising drug target in advanced Alzheimer's disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-pi interactions, as revealed by the solved crystal structure of its complex with human BChE. Additionally, compound 3 inhibits BChE ex vivo and is noncytotoxic. In vitro pharmacokinetic experiments show that compound 3 is highly protein bound, highly permeable, and metabolically stable. Finally, compound 3 crosses the blood-brain barrier, and it improves memory, cognitive functions, and learning abilities of mice in a scopolamine model of dementia. Compound 3 is thus a promising advanced lead compound for the development of drugs for alleviating symptoms of cholinergic hypofunction in patients with advanced Alzheimer's disease.
ESTHER : Kosak_2018_J.Med.Chem_61_119
PubMedSearch : Kosak_2018_J.Med.Chem_61_119
PubMedID: 29227101
Gene_locus related to this paper: human-BCHE

Title : Development of an in-vivo active reversible butyrylcholinesterase inhibitor - Kosak_2016_Sci.Rep_6_39495
Author(s) : Kosak U , Brus B , Knez D , Sink R , Zakelj S , Trontelj J , Pislar A , Slenc J , Gobec M , Zivin M , Tratnjek L , Perse M , Salat K , Podkowa A , Filipek B , Nachon F , Brazzolotto X , Wieckowska A , Malawska B , Stojan J , Rascan IM , Kos J , Coquelle N , Colletier JP , Gobec S
Ref : Sci Rep , 6 :39495 , 2016
Abstract : Alzheimer's disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.
ESTHER : Kosak_2016_Sci.Rep_6_39495
PubMedSearch : Kosak_2016_Sci.Rep_6_39495
PubMedID: 28000737
Gene_locus related to this paper: human-BCHE

Title : Galantamine as a preventive of diisopropylphosphorofluoridate toxicity effects in rat brain - Saghafi_2013_Folia.Biol.(Praha)_59_32
Author(s) : Saghafi MM , Zivin M , Pregelj P
Ref : Folia Biol (Praha) , 59 :32 , 2013
Abstract : Diisopropylfluorophosphate exerts its toxic effect by irreversibly inhibiting acetylcholinesterase. This results in over-stimulation of central and peripheral cholinergic activity. The aim of the present study was to evaluate the possible preventive effects of acute treatment with reversible acetylcholinesterase inhibitor galantamine against the signs of cholinergic toxic syndrome provoked by diisopropylfluorophosphate, such as hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance in a rat model of intoxication. The effects of these two anticholinesterases on acetylcholinesterase activity and on the expression of mRNA of the immediate early response gene c-fos in the brain were assessed by histochemical acetylcholinesterase staining and by in situ hybridization, respectively. Diisopropylfluorophosphate induced rapidly progressing hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance. The increased cholinergic cortical and hippocampal activity due to irreversible acetylcholinerase inhibition were indicated by the increased c-fos mRNA autoradiographic signal and by the inhibition of acetylcholinesterase staining, respectively. Galantamine by itself provoked transient and relatively weak inhibition of the acetylcholinesterase staining, while it did not induce increased c-fos mRNA expression or significant behavioural signs of cholinergic toxicity. Galantamine significantly reduced the rate of the onset, but not the maximal hypothermia induced by diisopropylfluorophosphate. Importantly, all the above-mentioned behavioural and neurochemical effects of diisopropylfluorophosphate were significantly reduced by galantamine. These results indicate that the acute pre-treatment with galantamine may have prophylactic effects against the intoxication by diisopropylfluorophosphate.
ESTHER : Saghafi_2013_Folia.Biol.(Praha)_59_32
PubMedSearch : Saghafi_2013_Folia.Biol.(Praha)_59_32
PubMedID: 23537526

Title : Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer's disease - Tratnjek_2013_Croat.Med.J_54_419
Author(s) : Tratnjek L , Zivin M , Glavan G
Ref : Croat Med J , 54 :419 , 2013
Abstract : AIM: To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer's disease pathogenesis and to define the cell types containing increased levels of Syt IV in the beta-amyloid plaque vicinity.
METHODS: Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex.
RESULTS: Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions).
CONCLUSIONS: Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer's disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.
ESTHER : Tratnjek_2013_Croat.Med.J_54_419
PubMedSearch : Tratnjek_2013_Croat.Med.J_54_419
PubMedID: 24170720

Title : Differential patterns of synaptotagmin7 mRNA expression in rats with kainate- and pilocarpine-induced seizures - Glavan_2012_PLoS.One_7_e36114
Author(s) : Glavan G , See RE , Zivin M
Ref : PLoS ONE , 7 :e36114 , 2012
Abstract : Previous studies in rat models of neurodegenerative disorders have shown disregulation of striatal synaptotagmin7 mRNA. Here we explored the expression of synaptotagmin7 mRNA in the brains of rats with seizures triggered by the glutamatergic agonist kainate (10 mg/kg) or by the muscarinic agonist pilocarpine (30 mg/kg) in LiCl (3 mEq/kg) pre-treated (24 h) rats, in a time-course experiment (30 min-1 day). After kainate-induced seizures, synaptotagmin7 mRNA levels were transiently and uniformly increased throughout the dorsal and ventral striatum (accumbens) at 8 and 12 h, but not at 24 h, followed at 24 h by somewhat variable upregulation within different parts of the cerebral cortex, amigdala and thalamic nuclei, the hippocampus and the lateral septum. By contrast, after LiCl/pilocarpine-induced seizures, there was a more prolonged increase of striatal Synaptotagmin7 mRNA levels (at 8, 12 and 24 h), but only in the ventromedial striatum, while in some other of the aforementioned brain regions there was a decline to below the basal levels. After systemic post-treatment with muscarinic antagonist scopolamine in a dose of 2 mg/kg the seizures were either extinguished or attenuated. In scopolamine post-treated animals with extinguished seizures the striatal synaptotagmin7 mRNA levels (at 12 h after the onset of seizures) were not different from the levels in control animals without seizures, while in rats with attenuated seizures, the upregulation closely resembled kainate seizures-like pattern of striatal upregulation. In the dose of 1 mg/kg, scopolamine did not significantly affect the progression of pilocarpine-induced seizures or pilocarpine seizures-like pattern of striatal upregulation of synaptotagmin7 mRNA. In control experiments, equivalent doses of scopolamine per se did not affect the expression of synaptotagmin7 mRNA. We conclude that here described differential time course and pattern of synaptotagmin7 mRNA expression imply regional differences of pathophysiological brain activation and plasticity in these two models of seizures.
ESTHER : Glavan_2012_PLoS.One_7_e36114
PubMedSearch : Glavan_2012_PLoS.One_7_e36114
PubMedID: 22567130

Title : Donepezil inhibits diisopropylfluorophosphate-induced seizures and up-regulation of synaptotagmin 4 mRNA - Saghafi_2010_Folia.Biol.(Praha)_56_256
Author(s) : Saghafi MM , Pregelj P , Zivin M
Ref : Folia Biol (Praha) , 56 :256 , 2010
Abstract : Reversible acetylcholinesterase inhibitor donepezil displays prophylactic effects against intoxication with irreversible organophosphorous acetylcholinesterase inhibitors. We used behavioural observation of yawning and epileptic seizures, histochemical acetylcholinesterase staining, and in situ hybridization of the immediate early genes, c-fos and synaptotagmin 4 (Syt4) mRNAs in the brain, to evaluate whether donepezil could protect the brain against the effects of the organophosphate anticholinesterase, diisopropylfluorophosphate, in a rat model of intoxication. Diisopropylfluorophosphatetreated animals exhibited frequent yawning, significant inhibition of acetylcholinesterase staining and upregulation of c-fos mRNA, but not the epileptic seizures or significant change of Syt4 mRNA levels. In order to reduce the threshold for the induction of cholinergic seizures, additional groups of rats were pre-treated with LiCl 24 h before the treatment with diisopropylfluorophosphate. These rats exhibited the seizures, a significant inhibition of acetylcholinesterase staining and significant upregulation of c-fos and Syt4 mRNA levels. All the above-mentioned effects of diisopropylfluorophosphate were inhibited by donepezil pre-treatment. Donepezil pre-treatment by itself induced only a comparatively weaker inhibition of acetylcholinesterase staining and infrequent yawning. We conclude that donepezil protects the brain against diisopropylfluorophosphate-induced effects and that Syt4 mRNA upregulation may serve as a novel marker for organophosphate-induced seizures.
ESTHER : Saghafi_2010_Folia.Biol.(Praha)_56_256
PubMedSearch : Saghafi_2010_Folia.Biol.(Praha)_56_256
PubMedID: 21324267

Title : Cholinergic regulation of striatal Nova mRNAs - Jelen_2010_Neurosci_169_619
Author(s) : Jelen N , Ule J , Zivin M
Ref : Neuroscience , 169 :619 , 2010
Abstract : Alternative splicing is an important mechanism for expanding proteome diversity from a limited number of genes, especially in higher vertebrates. Brain-specific splicing factors play an important role in establishing specific patterns of alternative splicing in the brain and thereby contribute to its complex architecture and function. Nova proteins are splicing factors that are expressed specifically in the central nervous system, where they regulate a large number of pre-mRNAs encoding synaptic proteins that are important for the balance of neuronal excitation and inhibition. Since this balance is interrupted in epileptic seizures, we explored whether LiCl/pilocarpine- or kainate-induced epileptic seizures would induce changes in the levels of Nova mRNAs in the rat brain. We found that the muscarinic agonist, pilocarpine, but not the glutamatergic agonist, kainate, induced a significant downregulation of Nova2 mRNA and upregulation of all three Nova1 mRNA isoforms in the striatum. Treatment with the muscarinic antagonist, scopolamine, at the onset of pilocarpine-induced seizures inhibited the seizures and the changes in Nova mRNA levels. Therefore it seems likely that pilocarpine stimulation of muscarinic acetylcholine receptors was a prerequisite for the observed changes, while the contribution of other striatal neurotransmitter systems activated by seizures could not be excluded. We propose that the LiCl/pilocarpine seizure model could serve as a valuable tool for studying mechanisms of Nova-regulated alternative splicing in rat striatum.
ESTHER : Jelen_2010_Neurosci_169_619
PubMedSearch : Jelen_2010_Neurosci_169_619
PubMedID: 20470870

Title : Synaptotagmins in neurodegeneration - Glavan_2009_Anat.Rec.(Hoboken)_292_1849
Author(s) : Glavan G , Schliebs R , Zivin M
Ref : Anatomical Record (Hoboken) , 292 :1849 , 2009
Abstract : Synaptotagmins (Syts) are transmembrane proteins involved in the regulation of membrane trafficking. Here, we summarize literature data that provide growing evidence that several Syts are involved in the pathophysiological mechanisms of temporal lobe epilepsy and Parkinson's disease, as well as few reports related to brain ischemia and Alzheimer's disease (AD). We also report new data from our laboratories, showing changes of the expression of several Syts in Tg2576 mouse model of AD that may be related to neuroinflammation surrounding the beta-amyloid plaques. Furthermore, we demonstrate N-methyl-D-aspartate receptor-mediated upregulation of Syt 4 mRNA in a model of excitotoxic striatal lesion induced by unilateral striatal injection of quinolinic acid, associating the upregulation of Syt 4 with mechanisms of excitotoxicity. We propose that pharmacological manipulation of Syt expression in animal models of neurodegeneration should be further explored, as it may help to clarify the role of individual Syt isoforms in the regulation of membrane trafficking in neurodegeneration.
ESTHER : Glavan_2009_Anat.Rec.(Hoboken)_292_1849
PubMedSearch : Glavan_2009_Anat.Rec.(Hoboken)_292_1849
PubMedID: 19943339

Title : Prolonged treatment with donepezil increases acetylcholinesterase expression in the central nervous system - Zivin_2008_Psychiatr.Danub_20_168
Author(s) : Zivin M , Pregelj P
Ref : Psychiatr Danub , 20 :168 , 2008
Abstract : OBJECTIVE: Acetylcholinesterase (AChE), an enzyme catalysing rapid hydrolysis of acetylcholine is the major enzyme in the metabolism of this neurotransmitter in the central nervous system and in the skeletal and smooth muscles. Donepezil is a reversible, primarily non-competitive, selective inhibitor of AChE used in patients with Alzheimer's disease for the improvement of cognitive deficits. We hypothesized that prolonged treatment with donepezil could increase AChE mRNA levels and AChE activity in the central nervous system.
METHODS: The levels of AChE mRNA and AChE activity in the brain sections of 6 animals treated for 28 days with donepezil (2 mg/kg s.c.) were visualized by an autoradiographic method of in situ hybridization and by Koelle histochemical staining, respectively, and compared with 6 control animals treated with physiologic saline. The images of autoradiograms and of AChE-stained brain sections were densitometrically analysed with a computerized imaging analysis system.
RESULTS: We observed that 28-day treatment with donepezil in comparison to control treatment increased hippocampal AChE mRNA levels and AChE activity.
CONCLUSIONS: Our data suggest that AChE up-regulation induced by prolonged treatment with AChE inhibitors may be the rationale for up-titration of AChE inhibitors during the treatment of AD. Further preclinical and clinical data are needed to evaluate the relative impact of the up-regulation of AChE activity on the outcome of prolonged treatment of AD patients with donepezil.
ESTHER : Zivin_2008_Psychiatr.Danub_20_168
PubMedSearch : Zivin_2008_Psychiatr.Danub_20_168
PubMedID: 18587286

Title : Evolution of Nova-dependent splicing regulation in the brain - Jelen_2007_PLoS.Genet_3_1838
Author(s) : Jelen N , Ule J , Zivin M , Darnell RB
Ref : PLoS Genet , 3 :1838 , 2007
Abstract : A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.
ESTHER : Jelen_2007_PLoS.Genet_3_1838
PubMedSearch : Jelen_2007_PLoS.Genet_3_1838
PubMedID: 17937501

Title : Effect of apomorphine on striatal synaptotagmin 7 mRNA levels in reserpinized rats - Pal_2007_Neurosci.Lett_424_194
Author(s) : Pal R , Zivin M , Milutinovic A , Jernej B , Glavan G
Ref : Neuroscience Letters , 424 :194 , 2007
Abstract : Synaptotagmin 7 (Syt 7) is a Ca2+ sensor implicated in the regulation of membrane fusion in vesicular transport, but its precise role in neurons is still a matter of controversy. Dopaminergic drugs have been shown to modulate its expression in the striatum. Here we investigate whether dopamine receptor agonist-up-regulation of Syt 7 mRNA is specifically involved in the pathophysiological adaptations of hypersensitive striatum by analyzing other dopaminergic neurons containing brain regions. We treated rats with systemic reserpine injections that rapidly depletes dopamine throughout the brain, but leaves dopaminergic neurons spared from destruction. We analyzed the effects of apomorphine, a D1 and D2 receptor agonist on Syt 7 mRNA expression in caudate putamen, nucleus accumbens, cingulate cortex, substantia nigra compacta, ventral tegmental area and hippocampus. The treatment with reserpine resulted in akinesia, catalepsy and rigidity and up-regulation of proenkephalin and down-regulation of preprotachykinin mRNA in caudate putamen, indicating a severe depletion. By acute treatment with apomorphine proenkephalin mRNA was down-regulated and preprotachykinin mRNA up-regulated in the caudate putamen of reserpinized rats. Apomorphine increased Syt 7 mRNA levels only in striatum (caudate putamen and nucleus accumbens) of reserpinized rats, while in other brain regions it did not have such effect. The reserpinization and/or apomorphine treatment had no effect on Syt 1 mRNA expression in caudate putamen. It may be concluded, that in the striatum depleted of biogene amines, such as occurs after reserpine treatment, the up-regulation of Syt 7 could play a specific role as part of hypersensitive response to dopaminergic agonists.
ESTHER : Pal_2007_Neurosci.Lett_424_194
PubMedSearch : Pal_2007_Neurosci.Lett_424_194
PubMedID: 17719177

Title : Upregulation of synaptotagmin IV protein in kainate-induced seizures - Glisovic_2007_Neuroreport_18_831
Author(s) : Glisovic S , Glavan G , Saghafi MM , Zivin M
Ref : Neuroreport , 18 :831 , 2007
Abstract : Synaptotagmin IV is a product of immediate early-response gene. It is involved in the regulated neurosecretion in the brain. Its putative role, however, in vesicular transport and localization in secretor y vesicles is still a matter of debate. Here we followed the spatiotemporal pattern of synaptotagmin IV protein upregulation in the hippocampus, caudate putamen, nucleus accumbens, nucleus amygdalae, piriform and entorhinal cortices of rats with kainate-induced seizures. We found that upregulation pattern paralleled the direction of depolarization through the hippocampus and also reflecting seizure activity spreading to other brain regions. We speculate that synaptotagmin IV may have a role in the vesicular transport of the upregulated peptides and proteins involved in the plasticity and/or neurodegeneration provoked by the kainate.
ESTHER : Glisovic_2007_Neuroreport_18_831
PubMedSearch : Glisovic_2007_Neuroreport_18_831
PubMedID: 17471076

Title : Differential expression of striatal synaptotagmin mRNA isoforms in hemiparkinsonian rats - Glavan_2005_Neurosci_135_545
Author(s) : Glavan G , Zivin M
Ref : Neuroscience , 135 :545 , 2005
Abstract : Synaptotagmins (Syts) constitute a multi-gene family of 15 putative membrane trafficking proteins. The expression of some of the Syts in the brain might be dopaminergically controlled and thus affected by dopamine depletion in Parkinson's disease. We used hemiparkinsonian rats to investigate the effects of chronic striatal dopamine depletion and the acute effects of antiparkinsonic drug L-DOPA or D1 agonist (+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF82958) on the levels of striatal Syt I, II, IV, VI, VII, X, XI mRNA isoforms. On the 6-hydroxydopamine (6-OHDA)-lesioned side we observed a nearly total loss of tyrosine hydroxylase (TH), synaptotagmin I, Syt IV, Syt VII and Syt XI mRNA levels in the substantia nigra compacta (SNc). In dopamine-depleted striatum we also found a significant down-regulation Syt II and up-regulation of Syt X mRNA levels that could not be reversed by the acute treatment either with L-DOPA or SKF82958. By contrast, these two drugs induced an increase of Syt IV and Syt VII mRNA levels. A time-course study revealed the highest levels of Syt IV and VII mRNAs to occur at two hours and 12 hours after the treatment with SKF82958, respectively. D1 antagonist (+/-)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) but not D2 antagonist haloperidol prevented the L-DOPA-driven increase of Syt IV and VII mRNAs. These results imply that synaptic plasticity in response to chronic striatal dopamine depletion involves a complex pattern of changes in striatal Syt mRNA expression. The L-DOPA treatment does not reverse the changes in Syt II and Syt X gene expression, but recruits additional, D1 receptor-mediated changes in Syt IV and Syt VII gene expression. Whether these D1 receptor-mediated changes play a role in the alterations of synaptic transmission that results in the unwanted side effects of chronic L-DOPA treatment in Parkinson's disease remains to be determined.
ESTHER : Glavan_2005_Neurosci_135_545
PubMedSearch : Glavan_2005_Neurosci_135_545
PubMedID: 16111820

Title : N-tert-butyl-alpha-phenylnitrone, a free radical scavenger with anticholinesterase activity does not improve the cognitive performance of scopolamine-challenged rats - Milivojevic_2001_Int.J.Dev.Neurosci_19_319
Author(s) : Milivojevic N , Babic K , Milatovic D , Dettbarn WD , Sket D , Zivin M
Ref : Int J Developmental Neuroscience , 19 :319 , 2001
Abstract : Reversible inhibitors of acetylcholinesterase improve spatial learning and memory in animal models of cognitive impairment. Here we investigate if the beneficial effects of free radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on cognitive performance could be explained by its recently discovered anticholinesterase activity. Morris water maze experiment was performed to examine the effect of PBN on the impairment of spatial learning and memory induced by the antagonist of cholinergic muscarinic transmission scopolamine. In situ hybridization histochemistry experiment was performed to study its effects on the induction of immediate early gene expression (c-fos, c-jun) by dopamine D1 receptor agonist SKF-82958 and on the augmentation of the SKF-82958-induced expression of these genes by scopolamine. In both experiments, the effects of PBN were compared to the effects of reversible anticholinesterase physostigmine. We found that physostigmine but not PBN significantly reversed the cognitive impairment in scopolamine-challenged rats, prevented the induction of c-fos and c-jun mRNAs by SKF-82958 and attenuated the augmentation of the SKF-82958-induced expression of these genes by scopolamine. The present experiments did not reveal a significant in vivo anticholinesterase activity of PBN.
ESTHER : Milivojevic_2001_Int.J.Dev.Neurosci_19_319
PubMedSearch : Milivojevic_2001_Int.J.Dev.Neurosci_19_319
PubMedID: 11337201

Title : Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase - Milatovic_2000_Free.Radic.Biol.Med_28_597
Author(s) : Milatovic D , Radic Z , Zivin M , Dettbarn W
Ref : Free Radic Biol Med , 28 :597 , 2000
Abstract : N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.
ESTHER : Milatovic_2000_Free.Radic.Biol.Med_28_597
PubMedSearch : Milatovic_2000_Free.Radic.Biol.Med_28_597
PubMedID: 10719241

Title : Spin trapping agent phenyl-N-tert-butylnitrone prevents diisopropylphosphorofluoridate-induced excitotoxicity in skeletal muscle of the rat - Milatovic_2000_Neurosci.Lett_278_25
Author(s) : Milatovic D , Zivin M , Hustedt E , Dettbarn WD
Ref : Neuroscience Letters , 278 :25 , 2000
Abstract : Indirect evidence suggests that reactive oxygen species (ROS) may mediate muscle fiber necrosis following muscle hyperactivity induced by the anticholinesterase diisopropylphosphorofluoridate (DFP). Pronounced muscle fasciculations and muscle fiber necrosis were seen when acetylcholinesterase (AChE) activity was reduced to less than 30% of control. The spin trapping agent phenyl-N-tert-butylnitrone (PBN) was used in vivo to directly assess the formation of ROS during DFP (1.75 mg/kg, s.c.) induced muscle hyperactivity. Pretreatment with PBN (300 mg/kg, i.p.), the concentration necessary for in vivo spin trapping, prevented muscle hyperactivity as well as necrosis and attenuated the DFP induced AChE inhibition otherwise seen in DFP only treated rats. PBN had no effect when given after fasciculations were established. Muscle extracts from PBN and DFP treated rats subjected to electron spin resonance (ESR) spectroscopy tested negative for ROS. While the role of PBN as an antioxidant is well established, its prophylactic effect against excitotoxity induced by an AChE inhibitor are due to its protection of AChE, an unexpected non-antioxidant action.
ESTHER : Milatovic_2000_Neurosci.Lett_278_25
PubMedSearch : Milatovic_2000_Neurosci.Lett_278_25
PubMedID: 10643792

Title : Nitrone spin trapping compound N-tert-butyl-alpha-phenylnitrone prevents seizures induced by anticholinesterases - Zivin_1999_Brain.Res_850_63
Author(s) : Zivin M , Milatovic D , Dettbarn WD
Ref : Brain Research , 850 :63 , 1999
Abstract : The neuroprotection afforded by spin trapping agents such as N-tert-butyl-alpha-phenylnitrone (PBN) has lent support to the hypothesis that increased production of reactive oxygen species (ROS) is a major contributing factor to excitotoxicity, aging and cognitive decline. Little is known, however, about the pharmacological properties of PBN. We have compared the acute effects of PBN on the development of seizures induced by the irreversible acetylcholinesterase (AChE) inhibitor diisopropylphosphorofluoridate (DFP), the reversible AChE inhibitor physostigmine (PHY), the muscarinic cholinergic receptor agonist pilocarpine (PIL) and the glutamatergic receptor agonist kainic acid (KA). Rats were sacrificed 90 min after the injection of seizure-inducing agents. In situ hybridization was used to detect the induction of immediate early gene (IEG) c-fos and c-jun mRNA's and the levels of AChE mRNA. The activity of AChE was visualized by AChE staining and quantified using an in vitro AChE assay. The seizures correlated with the induction of IEG mRNA's with all agents used. The pre-treatment with 150 mg/kg of PBN prevented DFP- and PHY-induced seizures and the related expression of IEG mRNA's, but had no effect on PIL- or KA-induced seizures and associated IEG mRNA's changes. PBN prevented seizures and significantly protected AChE activity against DFP inhibition when given before, but not when given after DFP. This study shows that PBN specifically protects against anticholinesterase-induced seizures by reversible protection of AChE activity and not by the blockade of muscarinic or glutamate receptors, reactivation of AChE or scavenging of ROS. The anticholinesterase properties should be considered when using PBN in studies of cholinergic dysfunction.
ESTHER : Zivin_1999_Brain.Res_850_63
PubMedSearch : Zivin_1999_Brain.Res_850_63
PubMedID: 10629749

Title : Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat\; comparison to the radioactive technique - Kreft_1996_Pflugers.Arch_431_R309
Author(s) : Kreft S , Zajc-Kreft K , Zivin M , Sket D , Grubic Z
Ref : Pflugers Arch , 431 :R309 , 1996
Abstract : In this preliminary report nonradioactive digoxigenine-based and radioactive in situ hybridization procedures for the localization of acetylcholinesterase mRNA were tested and compared in rat brain. General patterns of Ache mRNA localization observed by both techniques did not differ significantly and were practically the same as reported in previous in situ studies on the mammalian brain. Shorter procedure time and avoidance of precautions necessary at work with radioactive materials are major advantages of nonradioactive technique. Under- and over- staining can be prevented by direct examination of coloring reaction. Faint staining in the control experiment with heterologous DNA suggests that proper stringency is essential for the specificity of staining.
ESTHER : Kreft_1996_Pflugers.Arch_431_R309
PubMedSearch : Kreft_1996_Pflugers.Arch_431_R309
PubMedID: 8739388