Fractions Of Brain Homogenates Research Articles

Hierarchical involvement of molecular players in human neocortex in the course of preclinical and symptomatic Alzheimer’s disease

AbstractBackgroundProteomic profile changes have been reported in the brains from individuals having Alzheimer’s disease. However, it is not yet clear whether they represent a continuous process from mild to severe changes, or whether the proteins become involved step‐by‐step in a hierarchal manner. Additionally, in previous studies, cases with initial Aβ plaque and NFT pathology were considered as controls, and brain homogenates were not separated into distinct fractions. Therefore, there is a gap of knowledge regarding the initial steps of AD pathogenesis and the spatial resolution of the respective protein changes in terms of subcellular distribution.MethodWe analyzed soluble, dispersible (insoluble proteins and vesicles in mixtures with the intra‐/extracellular fluid), membrane‐associated (SDS‐soluble), and plaque‐associated (formic‐acid‐soluble) fractions of brain homogenates from 19 control cases without AD pathology, 17 pathologically preclinical AD (p‐preAD), and 17 symptomatic AD (symAD) cases by label‐free liquid chromatography coupled to tandem mass spectrometry. Proteomic changes and related pathways involved in p‐preAD and symAD were determined by integrated analyses combining quantitative proteomics, neuropathological measurements, regression analysis, and gene ontology overrepresentation tests.ResultThree hierarchical classes of proteins, namely early‐ and late‐responding proteins and gradually changing proteins were identified across control, p‐preAD and symAD samples. Additionally, five proteins exhibited reduced abundance in the soluble fraction accompanied by increase in the membrane‐associated fraction between symAD and the group of p‐preAD + control cases, suggesting pathological alterations in their functions. Gene ontology analysis showed that the synaptic vesicle cycle and other endocytosis‐related pathways were overrepresented by early‐responding and gradually changing proteins, whereas caspase‐mediated cleavage of cytoskeletal proteins were enriched in the late‐responding protein class.ConclusionWe identified an apparent hierarchy of key proteins in the human brain during AD pathogenesis, from which the early responding proteins point to pathways that are not yet a main focus in AD research. This hierarchy of proteome change in the neocortex clearly distinguished the preclinical phase from the symptomatic phase and identified the synaptic vesicle cycle as a likely primary affected function in AD initiation. Our results may open new roads for therapies by intervening on those key proteins and their respective pathways.

Анализ связывающей способности сигма-1 рецепторов при эмоционально-стрессовом воздействии и введении анксиолитика афобазола

Специфическое связывание лиганда сигма-1 рецепторов (Sigma1R) [3H](+)-пентазоцина в Р2 и Р3 фракциях гомогенатов головного мозга определяли у инбредных мышей BALB/с и C57Bl/6 после эмоционально-стрессового воздействия в тесте «Открытое поле» (ОП) и аутбредных мышей CD-1 после введения анксиолитика афобазола. Установлено, что экспозиция в ОП увеличивает специфическое связывание [3H](+)-пентазоцина в Р3 фракции гомогенатов головного мозга мышей BALB/с, характеризующихся реакцией замирания в тесте ОП, и не влияет на связывание лиганда у мышей C57Bl/6 с активным поведением в тесте. Афобазол в дозе 5 мг/кг вызывает увеличение специфического связывания [3H](+)-пентазоцина в Р2 фракции гомогенатов головного мозга аутбредных мышей CD-1. Полученные данные свидетельствуют об участии Sigma1R в механизмах формирования реакций на эмоционально-стрессовое воздействие и в фармакодинамике афобазола.

The APOE ε4 Allele Is Associated with Lower Selenium Levels in the Brain: Implications for Alzheimer's Disease.

The antioxidant activity of selenium, which is mainly conferred by its incorporation into dedicated selenoproteins, has been suggested as a possible neuroprotective approach for mitigating neuronal loss in Alzheimer's disease. However, there is inconsistent information with respect to selenium levels in the Alzheimer's disease brain. We examined the concentration and cellular compartmentalization of selenium in the temporal cortex of Alzheimer's disease and control brain tissue. We found that Alzheimer's disease was associated with decreased selenium concentration in both soluble (i.e., cytosolic) and insoluble (i.e., plaques and tangles) fractions of brain homogenates. The presence of the APOE ε4 allele correlated with lower total selenium levels in the temporal cortex and a higher concentration of soluble selenium. Additionally, we found that age significantly contributed to lower selenium concentrations in the peripheral membrane-bound and vesicular fractions. Our findings suggest a relevant interaction between APOE ε4 and selenium delivery into brain, and show changes in cellular selenium distribution in the Alzheimer's disease brain.

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.

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.

Different CSF β-amyloid processing in Alzheimer’s and Creutzfeldt–Jakob disease

Decreased levels of β-amyloid (Aβ) 1-42 in cerebrospinal fluid (CSF) are characteristic for Alzheimer's disease (AD) and are also evident in Creutzfeldt-Jakob disease (CJD). Aβ plaques are thought to be responsible for this decrease in AD patients, whereas such Aβ plaques are rarely seen in CJD. To investigate the Aβ pattern in brain and CSF of neuropathologically confirmed CJD and AD patients we used an electrophoretic method to investigate Aβ peptide fractions which are not accessible to ELISA and immunohistochemistry. We analyzed Aβ peptides in the CSF of autopsy-confirmed CJD and AD patients and the corresponding brain homogenates using a quantitative urea-based Aβ electrophoresis immunoblot (Aβ-SDS-PAGE/immunoblot).The CSF Aβ1-42 decrease correlated with the brain Aβ load in AD, but not in CJD. There was no difference in the soluble fractions of brain homogenate in AD and CJD. We therefore conclude that different mechanisms in AD and CJD are responsible for the Aβ1-42 decrease in the CSF.

The Therapeutic Effects of the Herbal Medicine, Juzen-taiho-to, on Amyloid-β Burden in a Mouse Model of Alzheimer's Disease

Innate immunity, especially that involving macrophage function, reportedly diminishes with advancing age and in patients with Alzheimer's disease (AD). In this study, we tried to elicit the non-specific activation of peripheral macrophages by oral administration of the herbal medicine Juzen-taiho-to (JTT), to assess its effect as a possible treatment for AD patients. Amyloid-beta protein precursor transgenic mice were used as a model of AD to clarify the effect of JTT. Activated macrophages derived from bone marrow cross the blood-brain barrier, and then develop into microglia, which phagocytose aggregated amyloid-beta (Abeta) in senile plaques. Here we show that orally administered JTT increased the number of CD11b-positive ramified microglia in the mouse brain. The immunohistochemical examination of brain sections stained with polyclonal anti-Abeta antibody showed reduced Abeta burden, and Abeta levels were also decreased in the insoluble fractions of brain homogenates, as determined by ELISA. Thus, the activation of peripheral macrophages by JTT might be a potential new therapeutic strategy for AD.

Ixeris dentata extract maintains glutathione concentrations in mouse brain tissue under oxidative stress induced by kainic acid.

The neuroprotective effects of a water fraction from the methanol extract of Ixeris dentata (WFID) against oxidative stress in the brain of mice challenged with kainic acid were examined by evaluating behavioral characteristics and biochemical parameters of oxidative stress. Male ICR mice were divided into three groups: a control group that received no treatment and two groups challenged with kainic acid either with or without WFID (1.0 g/kg) for 4 consecutive days. On day 3 kainic acid (50 mg/kg) was intraperitoneally administered in the two challenged groups. When compared with the vehicle-treated control, no significant changes in body and brain weights were observed in mice administered WFID. Administration of kainic acid only caused a lethality of approximately 62.5%, and resulted in a significant decrease of total glutathione concentrations in the brain tissue. When WFID was investigated for neuroprotective action, WFID reduced the lethality (37.5%) of kainic acid, and the behavioral signs of its neurotoxicity. Moreover, the administration of WFID restored the glutathione concentrations in the cytosolic fraction of brain homogenate to control levels (P <.05). Furthermore, glutathione peroxidase activity was restored significantly (Plt;.05) in the cytosolic portion of brain homogenate, whereas glutathione reductase activity was not. These results suggest that I. dentata contains a functional agent that protects against oxidative stress in the brains of mice.

Increase in calcium content and Ca(2+)-ATPase activity in the brain of fasted rats: comparison with different ages.

The effect of fasting on calcium content and Ca(2+)-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca(2+)-ATPase activity in the microsomal and mitochondrial functions of the brain homogenate from young and elderly rats were significantly increased by overnight-fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca(2+)-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca(2+)-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration-induced increase in brain calcium content was greater in elderly rats than in young rats. Also, calcium administration caused a significant increase in Ca(2+)-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca(2+)-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting-induced increase in brain calcium content is involved in Ca(2+)-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy-dependent mechanism in brain calcium accumulation.

Stimulatory effect of fractions of brain homogenate on protein synthesis in ovaries of Eisenia fetida Sav. In Vitro (Oligochaeta, Annelida)

AbstractIn Eisenia fetida, during the period of genital activity, the synthesis of androgen by testicular tissues is under the control of a neurohormone elaborated by cerebral ganglia (CG). These ganglia also have a trophic effect on ovarian protein synthesis. Present results confirm that incorporation in vitro of [3H]‐leucine by ovaries is stimulated by a cerebral neurohormone. Furthermore, fractions of brain homogenate, obtained by gel filtration on Sephadex G 25, have the same action on ovaries in organ culture as CG. Two specific fractions (fractions 3 and 4) stimulate protein synthesis in the ovary. In the ovarian oocytes, the rate of vitellogenesis is low, since in cultured isolated ovaries the amount of radioactive precursor incorporated is only 0.06% of the amount added and this percentage remains low in the ovaries cultured with fractions 3 and 4 (0.15% and 0.21%, respectively) or with CG (0.24%). Further research is necessary to obtain and characterize the biologically active material. © 1993 Wiley‐Liss, Inc.

Isolation of a peptide initiating bursting activity in an identifiedHelix pomatia neuron

A peptide initiating bursting activity when applied to the soma of identified neuron RPal ofHelix pomatia (if such activity was absent) or increasing the amplitude of waves of membrane potential (if it was low), was isolated from the water-soluble fraction of brain homogenate. Application of the peptide and of the original material for its isolation (the water-soluble fraction of snail brain homogenate) evoked identical changes in the character of electrical activity of neuron RPal. It is concluded from the experimental results that the isolated protein possesses a specific action, qualitatively different from that of other known peptides, on this neuron and is the active principle of the modulating factor described previously. It is postulated that under natural conditions the modulating factor is secreted by an unidentified peptidergic interneuron in the region of axo-somatic synapses, evoking bursting activity in neuron RPal.

Effect of piperidines and fire ant venom on ATPase activities from brain homogenate fractions and characterization of Na +-K + ATPase inhibition

Effect of piperidines and fire ant venom on ATPase activities from brain homogenate fractions and characterization of Na +-K + ATPase inhibition

The effect of neonatal thyroidectomy on myelination in the rat brain

The deposition of lipids in rat brain has been studied after neonatal thyroidectomy. The amounts of phospholipids and cholesterol per g wet wt. of brain were unaffected during the first fortnight of life; they were reduced at 23 days, but approached the normal values at 35 days or later. On the other hand, a lasting and significant reduction was observed in the concentration of cerebrosides. The content of gangliosides was similar to the controls in the cerebrum of 23-day-old hypothyroid rats. The amounts of phospholipids and cholesterol in the cerebrum were somewhat reduced in the 35- and 43-day-old rats after neonatal thyroidectomy but the reduction was similar in scale to that observed in brain weigth. The amount of cerebroside was, however, reduced to a greater extent (40%). Confirmation that the effect of thyroid deprivation on the deposition of lipids was related to a decrease in the extent of myelination has come from experiments in which the myelin containing fraction of brain homogenates was isolated. The dry weight of myelin obtained from the brain of 43-day-old rats thyroidectomized at birth was reduced by more than 30%, but the lipid composition of myelin was unaffected. The yield of myelin per g brain assessed by determining the cholesterol content of the myelin fraction was also reduced in the 25- and 35-day-old hypothyroid rats, but the timing of myelination was apparently unaffected. Replacement therapy instituted from the time of thyroidectomy reversed completely the reduction in myelin deposition as assessed by the amount of cerebrosides per unit weight of cerebrum.

Excitatory and depressant properties of certain brain fractions.

ALTHOUGH it is generally believed that at most synapses in the brain ‘transmitter’ substances are released from nerve endings to excite or inhibit the post-synaptic cells, the identity of the transmitters involved is still very much in doubt. Gray and Whittaker1 have shown that a fraction of brain homogenates can be prepared containing mainly pinched-off nerve endings. More recently it has been possible to release apparently intact synaptic vesicles from such a nerve-ending fraction and to separate them from other membrane structures by density-gradient centrifugation2. The purified vesicle fraction contains acetylcholine but not the other components of the acetylcholine system. Since nerve endings can be expected to contain substantial amounts of various transmitter substances, we have examined the excitatory and depressant effects produced by various subtractions of guinea-pig brain, when applied from micropipettes to single units in the guinea-pig's cerebral cortex.