Brain Cytosol Research Articles

Alpha-Synuclein and GM1 Ganglioside Co-Localize in Neuronal Cytosol Leading to Inverse Interaction-Relevance to Parkinson's Disease.

Research on GM1 ganglioside and its neuroprotective role in Parkinson's disease (PD), particularly in mitigating the aggregation of α-Synuclein (aSyn), is well established across various model organisms. This essential molecule, GM1, is intimately linked to preventing aSyn aggregation, and its deficiency is believed to play a key role in the initiation of PD. In our current study, we attempted to shed light on the cytosolic interactions between GM1 and aSyn based on previous reports demonstrating gangliosides and monomeric aSyn to be present in neuronal cytosol. Native-PAGE and Western blot analysis of neuronal cytosol from mouse brains demonstrated the presence of both GM1 and monomeric aSyn in the neuronal cytosol of normal mouse brain. To demonstrate that an adequate level of GM1 prevents the aggregation of aSyn, we used NG108-15 and SH-SY5Y cells with and without treatment of 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), which inhibits the synthesis/expression of GM1. Cells treated with PPMP to reduce GM1 expression showed a significant increase in the formation of aggregated aSyn compared to untreated cells. We thus demonstrated that sufficient GM1 prevents the aggregation of aSyn. For this to occur, aSyn and GM1 must show proximity within the neuron. The present study provides evidence for such co-localization in neuronal cytosol, which also facilitates the inverse interaction revealed in studies with the two cell types above. This adds to the explanation of how GM1 prevents the aggregation of aSyn and onset of Parkinson's disease.

Prosaposin is a novel coenzyme Q10-binding protein.

Coenzyme Q10 (CoQ10) is essential for mitochondrial ATP production and functions as an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, and we previously described saposin B as a CoQ10-binding and transfer protein. Here, we report that prosaposin, the precursor of saposin B, also binds CoQ10. As prosaposin is both a secretory protein and integral membrane protein, it is ubiquitous in the body. Prosaposin was isolated from human seminal plasma, and CoQ10 was extracted from hexane solution into the water phase. It was additionally found that immunoprecipitates of mouse brain cytosol generated using two different anti-prosaposin antibodies contained coenzyme Q9. Furthermore, mouse liver cytosol and mouse kidney cytosol also contained prosaposin-coenzyme Q9 complex. These results suggest that prosaposin binds CoQ10 in human cells and body fluids. The significance and role of the Psap-CoQ10 complex in vivo is also discussed.

Metallobiochemistry of ultratrace levels of bismuth in the rat II. Interaction of 205+206Bi3+ with tissue, intracellular and molecular components

BackgroundKnowledge on Bi metabolism in laboratory animals refers to studies at “extreme” exposures, i.e. pharmacologically relevant high-doses (mg kg−1 b.w.) in relation to its medical use, or infinitesimal doses (pg kg−1b.w.) concerning radiobiology protection and radiotherapeutic purposes. There are no specific studies on metabolic patterns of environmental exposure doses (ultratrace level, μg kg−1 b.w.), becoming in this context Bi a “heavy metal fallen into oblivion”. We previously reported the results of the metabolic fate of ultratrace levels of Bi in the blood of rats [1]. In reference to the same study here we report the results of the retention and tissue binding of Bi with intracellular and molecular components. MethodsAnimals were intraperitoneally injected with 0.8 μg Bi kg−1 b.w. as 205+206Bi(NO)3, alone or in combination with 59Fe for the radiolabeling of iron proteins. The use of 205+206Bi radiotracer allowed the determination of Bi down to pg fg−1 in biological fluids, tissues, subcellular fractions, and biochemical components isolated by differential centrifugation, size exclusion chromatography, solvent extraction, precipitation, immunoprecipitation and dialysis. Main findingsAt 24 h post injection the kidney contained by far the highest Bi concentration (10 ng g−1 wt.w.) followed by the thymus, spleen, liver, thyroid, trachea, femur, lung, adrenal gland, stomach, duodenum and pancreas (0.1 to 1.3 ng g−1 wt.w.). Brain and testis showed smaller but consistently significant concentrations of the element (0.03 ng g−1 wt.w). Urine was the predominant route of excretion. Intracellularly, liver, kidney, spleen, testis, and brain cytosols displayed the highest percentages (35%–58%) of Bi of homogenates. Liver and testis nuclei were the organelles with the highest Bi content (24 % and 27 %). However, when the recovered Bi of the liver was recorded as percent of total recovered Bi divided by percent of total recovered protein the lysosomes showed the highest relative specific activity than in other fractions. In the brain subcellular fractions Bi was incorporated by neuro-structures with the protein and not lipidic fraction of the myelin retaining 18 % of Bi of the total homogenate. After the liver intra-subcellular fractionation: (i) 65 % of the nuclear Bi was associated with the protein fraction of the nuclear membranes and 35 % with the bulk chromatin bound to non-histone and DNA fractions; (ii) about 50 % of the mitochondrial Bi was associated with inner and outer membranes being the other half recovered in the intramitochondrial matrix; (iii) in microsomes Bi showed a high affinity (close to 90 %) for the membranous components (rough and smooth membranes); (iv) In the liver cytosol three pools of Bi-binding proteins (molecular size > 300 kDa, 70 kDa and 10 kDa) were observed with ferritin and metallothionein–like protein identified as Bi-binding biomolecules. Three similar protein pools were also observed in the kidney cytosol. However, the amount of Bi, calculated in percent of the total cytosolic Bi, were significantly different compared to the corresponding pools of the liver cytosol. ConclusionsAt the best of our knowledge the present paper represents the first in vivo study, on the basis of an environmental toxicology approach, aiming at describing retention and binding of Bi in the rat at tissue, intracellular and molecular levels.

Effects of Multiple Doses of Dichloroacetate on GSTZ1 Expression and Activity in Liver and Extrahepatic Tissues of Young and Adult Rats.

Glutathione transferase zeta 1 (GSTZ1), expressed in liver and several extrahepatic tissues, catalyzes dechlorination of dichloroacetate (DCA) to glyoxylate. DCA inactivates GSTZ1, leading to autoinhibition of its metabolism. DCA is an investigational drug for treating several congenital and acquired disorders of mitochondrial energy metabolism, including cancer. The main adverse effect of DCA, reversible peripheral neuropathy, is more common in adults treated long-term than in children, who metabolize DCA more quickly after multiple doses. One dose of DCA to Sprague Dawley rats reduced GSTZ1 expression and activity more in liver than in extrahepatic tissues; however, the effects of multiple doses of DCA that mimic its therapeutic use have not been studied. Here, we examined the expression and activity of GSTZ1 in cytosol and mitochondria of liver, kidney, heart, and brain 24 hours after completion of 8-day oral dosing of 100 mg/kg per day sodium DCA to juvenile and adult Sprague Dawley rats. Activity was measured with DCA and with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNPP), reported to be a GSTZ1-selective substrate. In DCA-treated rats, liver retained higher expression and activity of GSTZ1 with DCA than other tissues, irrespective of rodent age. DCA-treated juvenile rats retained more GSTZ1 activity with DCA than adults. Consistent with this finding, there was less measurable DCA in tissues of juvenile than adult rats. DCA-treated rats retained activity with EPNPP, despite losing over 98% of GSTZ1 protein. These data provide insight into the differences between children and adults in DCA elimination under a therapeutic regimen and confirm that the liver contributes more to DCA metabolism than other tissues. SIGNIFICANCE STATEMENT: Dichloroacetate (DCA) is one of few drugs exhibiting higher clearance from children than adults, after repeated doses, for reasons that are unclear. We hypothesized that juveniles retain more glutathione transferase zeta 1 (GSTZ1) than adults in tissues after multiple DCA doses and found this was the case for liver and kidney, with rat as a model to assess GSTZ1 protein expression and activity with DCA. Although 1,2-epoxy-3-(4-nitrophenoxy)propane was reported to be a selective GSTZ1 substrate, its activity was not reduced in concert with GSTZ1 protein.

Purification, characterization and identification of rat brain cytosolic tyrosine transaminase as glutamine Transaminase-K

The current study was undertaken to investigate the spectrum of tyrosine transaminases enzymes in a cytosolic fraction of rat brain and to specifically purify and characterize a previously identified cytosolic brain enzyme possessing tyrosine/glyoxylate transaminase activity. Based upon extensive biochemical and immunochemical characterization of purified brain tyrosine/glyoxylate transaminase, we concluded the purified enzyme is glutamine transaminase-K (EC 2.6.1.64). This conclusion was based on: 1.) a concurrent enrichment in the tyrosine/glyoxylate and glutamine/phenylpyruvate transaminase activities during purification, 2.) demonstration of a co-substrate specificity for amino acids and α-keto acids that was highly consistent with published information for glutamine transaminase-K, 3.) results from detailed kinetic analysis, 4.) glutamine was a potent inhibitor of in vitro tyrosine/glyoxylate transamination, 5.) biochemical characterization, including pH optimum of 8.5 and spectrophotometric analysis and 6.) immunoanalytical analysis using a specific antiserum to rat renal glutamine transaminase-k. In addition, immunochemical characterization of a crude soluble extract of whole brain suggests that the in vitro tyrosine transaminase activity for several different α-keto acid co-substrates likely reflect the activity of glutamine transaminase-K. In conclusion, this investigation confirmed the presence of multiple tyrosine transaminase enzymes in a cytosolic extract of rat brain. Moreover, we concluded glutamine transaminase-K represents a predominant cytosolic enzyme in rat brain that's capable of catalyzing in vitro transamination of p-tyrosine and other aromatic amino acids, including the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. The purified transaminase possesses a broad co-substrate specificity with preferential reactivity with α-keto acids derived from neutral aliphatic and aromatic amino acids. Lastly, we identified a heterogeneous regional distribution of tyrosine/glyoxylate transaminase (glutamine transaminase-K) in rat brain with a significantly higher level of in vitro activity in cerebellum.

A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1.

Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin. Biochemical fractionation followed by mass spectrometric analyses identifies the Eps15-homology domain-containing protein1 (EHD1) as a novel ATP-dependent membrane fission catalyst. Together, our approach establishes an experimental workflow for the discovery of novel membrane fission catalysts.

Beneficial Effects of Piperine in global cerebral ischemia/reperfusion model in irradiated rats

Stroke is the third cause of death and a leading cause of chronic disability all around the world. Inflammation and oxidative stress are common pathogenic pathways of brain injury induced by ischemia/reperfusion (I/R). This work aims to investigate the possible role of Piperine as antioxidant and anti-inflammatory in attenuating the cerebral ischemia/reperfusion (I/R)-induced brain injury in whole body γ-irradiated rats. Global cerebral ischemia was induced in both irradiated and non-irradiated male Wistar rats, and was followed by reperfusion. The beneficial effect of pretreatment with piperine (10 mg/kg, once daily p.o. for 15 days) was examined on cerebral ischemia-induced brain injury in 24 h pre-irradiated (6 Gy) male Wistar rats. I/R increased brain content of malondialdehyde (MDA) and decreased its glutathione (GSH) content and total antioxidant capacity (TAC). It also raised serum lactate dehydrogenase (LDH) activity and brain cytosolic calcium (Ca2+) level. Pre-treatment with piperine significantly attenuated brain injury in I/R irradiated rats by decreasing cytosolic brain calcium level and serum lactate dehydrogenase (LDH) activity. In addition, a significant decrease in nuclear factor kappa-B (NF-kB) was recorded in piperine treated group. These findings indicate that administration of piperine ameliorated most of the tested biochemical changes induced by I/R in irradiated rat brain and serum.

Effects of Src kinase inhibition on expression of pro-caspase-2 after brain hypoxia in a piglet animal model.

Caspase-2 has features of both initiator and effector caspases. Previously, we have shown that brain hypoxia-induced production of caspases 1, 3, 8, and 9 is Src kinase mediated, a nonreceptor intracellular family of kinases. The present study tests the hypothesis that hypoxia results in increased expression of caspase-2 and this effect is mediated by Src kinase. Two to three days old newborn piglets were subjected to normoxia, hypoxia (Hx, FiO2 7%), and Src kinase inhibition (using PP2, 1 mg/kg, intravenous), followed by 30 min of acute hypoxia (Hx+PP2). ATP and phosphocreatine were determined biochemically to verify energy molecule depletion in the hypoxic groups. The cytosolic brain function was isolated and a western blot analysis was carried out using an antibody specific for the caspase-2. The immune-complex band density was expressed as OD/mm. Caspase-2 expression was increased two-fold in the Hx group. After Src kinase inhibition followed by hypoxia, caspase-2 expression was similar to normoxia levels. We conclude that hypoxia results in increased expression of caspase-2 protein in the cytosolic fraction of the cerebral cortex of the newborn piglets. This increase is mediated by Src kinase.

Notch1 hallmarks fibrillary depositions in sporadic Alzheimer's disease.

BackgroundNotch1 signaling is a cellular cascade with a fundamental role from brain development to adult brain function. Reduction in Notch1 affects synaptic plasticity, memory and olfaction. On the other hand, Notch1 overactivation after brain injury is detrimental for neuronal survival. Some familial Alzheimer’s disease (FAD) mutations in Presenilins can affect Notch1 processing/activation. Others report that Notch1 is overexpressed in sporadic Alzheimer’s disease (AD). These works indicate that imbalances in Notch1 may be implicated in AD pathophysiology. In this study, we addressed whether Notch1 alteration can be considered a hallmark of AD.ResultsImmunohistochemical analysis of Notch1 on cortical and hippocampal tissue from post-mortem patients indicates an accumulation of Notch1 in plaque-like structures in the brain parenchyma of subjects with sporadic AD. Further analysis shows that displaced Notch1 is associated with fibrillary tangles/plaques. Biochemical validation confirms an accumulation of Notch1 in cytosolic brain fractions. This increase in protein is not accompanied with a raise in the Notch1 targets Hes1 and Hey1. Examination of the cerebrospinal fluid (CSF) indicates that the full length and truncations of the Notch1 protein are reduced in AD patients hinting at an accumulation in the brain parenchyma.ConclusionsOur research indicates that Notch1 is significantly displaced and accumulated in fibrillary structures in the susceptible hippocampal and cortical regions of sporadic AD patients. The dominant deposition of Notch1 in the brain parenchyma and its general signal reduction in neurons is consistent in all the AD patients analyzed and suggests that Notch1 may potentially be considered a novel hallmark of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0327-2) contains supplementary material, which is available to authorized users.

Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.

Molecular mechanism of endothelial and vascular aging: implications for cardiovascular disease

Western societies are aging due to an increasing life span, decreased birth rates, and improving social and health conditions. On the other hand, the prevalence of cardiovascular (CV) and cerebrovascular (CBV) diseases rises with age. Thus, in view of the ongoing aging pandemic, it is appropriate to better understand the molecular pathways of aging as well as age-associated CV and CBV diseases. Oxidative stress contributes to aging of organs and the whole body by an accumulation of reactive oxygen species promoting oxidative damage. Indeed, increased oxidative stress produced in the mitochondria and cytosol of heart and brain is a common denominator to almost all CV and CBV diseases. The mitochondrial adaptor protein p66(Shc) and the family of deacetylase enzymes, the sirtuins, regulate the aging process, determine lifespan of many species and are involved in CV diseases. GDF11, a member of TGFβ superfamily with homology to myostatin also retards the aging process via yet unknown mechanisms. Recent evidence points towards a promising role of this novel 'rejuvenation' factor in reducing age-related heart disease. Finally, telomere length is also involved in aging and the development of age-related CV dysfunction. This review focuses on the latest scientific advances in understanding age-related changes of the CV and CBV system, as well as delineating potential novel therapeutic targets derived from aging research for CV and CBV diseases.

Temporal Changes in Caspase-1 and Caspase-8 Activities Following Brain Hypoxia With and Without Src kinase Inhibition in a Piglet Animal Model.

The Src family kinases are a family of intracellular, non-receptor tyrosine kinases that are involved in a variety of cellular functions including the regulation of inflammation and apoptosis after brain hypoxia. Caspase-1 (C1) activates IL-1β through the formation of complex structures, the inflammasomes, while caspase-8 (C8) is part of the extrinsic apoptotic pathway. C8 has been found to directly activate the production of IL-1β. Previously, we observed that C1 and IL-1β are increased in the acute phase after hypoxia in the brain of piglets, but they follow a different pattern long term, with C1 remaining activated throughout the period of observation, while IL-1β returning to baseline at 15 days. Src kinase inhibition ameliorated the activation of C1 and IL-1β early, but did not appear to have any effect long term. Prompted by these findings, we assessed the changes that occur over time (1 h and 15 days) in C1 and C8 activities after brain hypoxia as well as the effect of pretreatment with a Src kinase inhibitor, PP2 on these biochemical markers. Enzymatic activities were determined by spectrophotometry with measurements of C1 and C8 in each cytosolic brain sample (N = 4 in each group). We found that C1 and C8 activities increase in the acute phase following hypoxia in the brain of newborn piglets, with C8 relatively more than C1 (C8/C1 ratio increased from 2:1 as baseline to 3:1 in hypoxia). Fifteen days after hypoxia C8/C1 ratio decreased to about 1:1. In piglets that were pretreated with a Src kinase selective inhibitor (PP2) and then subjected to hypoxia, the C8/C1 ratio early increase was not observed. Immediately after hypoxia C8 and C1 follow a similar pattern of increase while long term this appears to dissociate. We propose that following this experimental methodology, the previously observed IL-1β production after hypoxia might be associated with C8 rather than C1 and that Src kinase is involved in the above process.

Di-Ras2 Protein Forms a Complex with SmgGDS Protein in Brain Cytosol in Order to Be in a Low Affinity State for Guanine Nucleotides

The Ras family of small GTPases function in a wide variety of biological processes as "molecular switches" by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain – Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.

Oxidative state and oxidative metabolism in the brain of rats with adjuvant-induced arthritis

The purpose of the present study was to evaluate the oxidative status of the brain of arthritic rats, based mainly on the observation that arthritis induces a pronounced oxidative stress in the liver of arthritis rats and that morphological alterations have been reported to occur in patients with rheumatoid arthritis. Rats with adjuvant-induced arthritis were used. These animals presented higher levels of reactive oxygen species (ROS) in the total brain homogenate (25% higher) and in the mitochondria (+55%) when compared to healthy rats. The nitrite plus nitrate contents, nitric oxide (NO) markers, were also increased in both mitochondria (+27%) and cytosol (+14%). Arthritic rats also presented higher levels of protein carbonyl groups in the total homogenate (+43%), mitochondria (+69%) and cytosol (+145%). Arthritis caused a diminution of oxygen consumption in isolated brain mitochondria only when ascorbate was the electron donor. The disease diminished the mitochondrial cytochrome c oxidase activity by 55%, but increased the transmembrane potential by 16%. The pro-oxidant enzyme xanthine oxidase was 150%, 110% and 283% higher, respectively, in the brain homogenate, mitochondria and cytosol of arthritic animals. The same occurred with the calcium-independent NO-synthase activity that was higher in the brain homogenate (90%) and cytosol (122%) of arthritic rats. The catalase activity, on the other hand, was diminished by arthritis in all cellular fractions (between 30 and 40%). It is apparent that the brain of rats with adjuvant-induced arthritis presents a pronounced oxidative stress and a significant injury to lipids and proteins, a situation that possibly contributes to the brain symptoms of the arthritis disease.

Нитрозирующий стресс и апоптоз нейронов са1-зоны гиппокампа в условиях моделирования хронической алкогольной интоксикации: нейропротективные эффекты тиоцетама

Fulfilled researches showed that modeling of chronic alcohol intoxication by daily intragastric ethanol administration to non-pedigreed male white rats (first 10 days – 15% ethanol solution in dose 4 g/kg, next 10 days – 15% ethanol solution in dose 6 g/kg, then 10 days – 25% ethanol solution in dose 4 g/kg) leads to activation of nitrosorbidi stress reactions and initiation of neuro-apoptosis. Thus, as compared with healthy animals group, in rats’ brain after chronic ethanol administration it was revealed significant increase of nitro-tyrosine (nitrosorbidi stress marker) in cytosolic and mitochondrial fractions of brain homogenate against the background of revealing of apoptosis molecular markers in hippocampal CA1 zone neurons (fragmentation of neuron´s nuclei of hippocampal CA1 zone, increase of density and ratio of apoptotic- and destructive-changed cells) as well as deprivation of anti-apoptotic mechanisms (decrease of density of Bcl-2-positive neurons in hippocampal CA1 zone and decrease of Bcl-2 protein concentration in hippocampus). Subsequent 14-day intragastric administration of complex neuro-metabolic cerebro-protector Thiocetam in dose 100 mg/kg to the animals after chronic alcoholization showed significant protective effect on hippocampal CA1 zone neurons. Thus, Thiocetam administration resulted in decrease of nitrotyrosine levels in mitochondrion and in cytosol of brain of animals with chronic alcoholization as compared with untreated animals and with animals receiving Piracetam. Thiocetam administration to experimental animals led to significant decrease of density of apoptotic- and destructive-changed neurons of hippocampal CA1 zone and ratio of apoptotic-changed cells as compared with control group and animals receiving Piracetam. Course of treatment with Thiocetam increases significantly the density of Bcl-2-positive neurons in CA1 zone of hippocampus and significantly increases concentration of Bcl-2 in brain tissue of experimental animals as compared with control group and animals which received Piracetam according the same schedule. The increase of Bcl-2 protein expression in animals received Thiocetam testifies to the activation of anti-apoptotic defense of damaged neurons under the influence of this preparation. We suppose that one of the molecular mechanisms of neuro-protective effect of Thiocetam in chronic intoxication is the breaking of NO-depended mechanisms of neuro-apoptosis.

Tubulin binds to the cytoplasmic loop of TRESK background K⁺ channel in vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell.

A Role for an Hsp70 Nucleotide Exchange Factor in the Regulation of Synaptic Vesicle Endocytosis

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

Brain nucleoside recycling

The rate limiting reactions of nucleotide synthesis are modulated by intracellular fluctuations of nucleoside triphosphate concentrations. This topic has been mostly studied at the level of the de novo nucleotide synthesis from simple precursors. However, there are districts, such as brain, which rely more heavily on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides. This raises the following question: how do these districts maintain the right balance between the purine and pyrimidine pools? We believe that it is now safe to state that a cross talk exists between the extra- and intracellular metabolism of purine and pyrimidine nucleosides in the brain. The extracellular space is the major site of nucleoside generation through successive dephosphorylations of released triphosphates, whereas brain cytosol is the major site of multiple phosphorylations of uptaken nucleosides at their 5′-position. Modulation of both extracellular nucleoside generation by membrane bound ectonucleotidases, and intracellular nucleoside phosphorylation by cytosolic kinases might contribute to maintain the right extra- and intracellular purine and pyrimidine nucleotide balance in the brain.