Protein analysis in the rat auditory brainstem by two-dimensional gel electrophoresis and mass spectrometry

A catalogue of the protein repertoire of processing centres in the central auditory system would greatly foster our knowledge on the anatomical and functional properties of this sensory system. Towards this goal, we report on the first mapping study of the protein content in the superior olivary complex (SOC) and the inferior colliculus (IC) of the rat auditory brainstem. The protein content of these two structures was assessed by means of two-dimensional gel electrophoresis (2-DGE) and mass spectrometry. To do so, proteins were first separated into four fractions by differential centrifugation. For comparison, corresponding cerebellar fractions were also analysed. Immunoblot analysis revealed highly enriched microsomal and cytosolic fractions; the other two fractions were mixtures of various subcellular compartments. Separation of the 800 g pellets (enriched for nuclear and plasma membrane proteins) and the 100,000 g supernatants (enriched for cytosolic proteins) by 2-DGE yielded between 456 and 674 distinct protein spots after silver staining. The overall protein pattern of all three tissues was similar for a given fraction. Fifty prominent protein spots of the SOC cytosolic fraction were identified by mass spectrometry and yielded information on thirty different genes with various cellular functions, e.g. primary metabolism, cytoarchitecture, and signal transduction. Sequencing of eleven corresponding spots from the SOC and IC cytosolic fractions confirmed the great similarity between the two samples. The results of this analysis are part of a novel integrated database of the gene repertoire of the auditory brainstem (ID-GRAB), that is publicly available (http://www.id-grab.de).     

Glycoproteins of rat skeletal muscle sarcolemma: characterization by two-dimensional gel electrophoresis and effect of denervation

Sarcolemmal membrane glycoproteins from rat mixed, fast, and slow muscles were characterized by concanavalin A (ConA) binding following two-dimensional polyacrylamide gel electrophoresis (PAGE). Analysis of electrophoretic profiles revealed that although sarcolemmal membranes prepared from these muscle types contained common glycoprotein species, each had a distinct glycoprotein pattern. In sarcolemma from mixed muscle, three major classes of ConA binding glycoproteins could be distinguished: (i) an acidic species of 110,000-120,000 Da, pI 5.0 to 5.3 (CG-1, ConA binding glycoproteins-1); (ii) a group of highly charged isomers, ranging from 75,000 to 80,000 Da pI 5.2 to 8.2 (CG-2); and (iii) a group of charged isomers of predominantly acidic nature of approximately 50,000 Da pI 5.2 to 5.8 (CG-3). ConA bound exclusively to CG-1 in sarcolemma from a fast muscle (extensor digitorum longus muscle, EDL). In soleus muscle sarcolemma (slow fiber type) both CG-1 and CG-3 were readily detected but CG-2 was markedly diminished. ConA binding to slow muscle sarcolemma revealed as well a glycoprotein species of 66,000-70,000 Da, pI 4.3-5.1 (CG-4), which was unique to this fiber type and as such may be a specific marker for slow fiber type. Denervation had no significant effect on the properties of ConA binding to mixed or slow muscle sarcolemma but dramatically altered the ConA binding to fast muscle sarcolemma, specifically increasing binding to CG-2. These findings demonstrate that denervation differentially affects the metabolism of ConA binding glycoproteins in these muscle types.     

An investigation of experimental conditions for studying protein phosphorylation in micro-slices of rat brain by two-dimensional electrophoresis

Procedures are described for studying protein phosphorylation in 1 mm diameter micro-slices of rat brain tissue using two-dimensional electrophoresis as analytical tool. The activity of several protein phosphorylating systems, including a major system phosphorylating a 40 kDa substrate complex, was highly dependent on the procedures used for micro-slice preparation and on the Ca2+-content of the preparation medium. Under optimal conditions the pattern of phosphorylation observed in micro-slices closely resembled that obtained by in vivo labelling.     

应用DIGE测定帕金森病脑脊液中蛋白变化

目的 测定帕金森病(PD)脑脊液中蛋白的变化,为进一步探索PD的生物标记物提供线索.方法 采用荧光差异凝胶电泳技术分离并筛选PD和正常对照者脑脊液中差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)或串联质谱技术进行鉴定并分析.结果 共发现20个明显的差异蛋白点,其中11个点在PD中上调,9个点下调.共鉴定出8个蛋白质,其中有3个未知蛋白,均表现为下调.蛋白MYPT1出现明显下调,载脂蛋白原、脂蛋白发生明显上调,载脂蛋白A-I的一个异构体发生上调,一个异构体发生下调.结论 MYPT1与突触功能有关,载脂蛋白原、脂蛋白、载脂蛋白A-I与胆固醇代谢有关,这些蛋白与PD发生有一定关联,有可能成为PD的生物标记物.     

Specific protein expression in a rat model of early focal cerebral ischemia Fluorescent two-dimensional difference gel electrophoresis

BACKGROUND: The use of fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases.OBJECTIVE: To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion (MCAO), and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING: Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006.MATERIALS: 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) were purchased from Amersham Bioscience, Sweden.METHODS: Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES: At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with > 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS.RESULTS: Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were (1 758 ± 43) protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased (n = 7) and decreased (n = 6) expression (P< 0.05). MALDI-TOF-MS results revealed two differential protein spots: a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group (P < 0.05).CONCLUSION: Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.     

Effects of chronic haloperidol and clozapine treatment on AMPA and kainate receptor binding in rat brain

BACKGROUND: Alterations in AMPA and kainate receptor binding have been revealed in post-mortem schizophrenic brains. As most patients had been treated with antipsychotics, medication effects cannot be excluded as a possible explanation for these results. METHODS: Within the framework of this animal study, we investigated [3H]AMPA and [3H]kainate receptor binding in different rat brain regions following 6 months of oral treatment with either haloperidol (1.5 mg/kg/day) or clozapine (45 mg/kg/day). RESULTS: AMPA receptor binding was increased after haloperidol treatment in the striatum, nucleus accumbens, cingulate cortex, and insular cortex. Clozapine showed increased AMPA receptor binding only in the anterior cingulate cortex. Kainate receptor binding was increased by both drugs in all hippocampal subfields. CONCLUSIONS: This altered receptor binding may be related to beneficial neuroleptic effects and side effects. Furthermore, neuroleptic therapy may contribute to some of the post-mortem findings in the striatum in schizophrenia.     

Widespread expression of Fos protein induced by acute haloperidol administration in the rat brain

The effect of acute haloperidol administration on Fos protein expression was examined immunohistochemically in discrete regions of the rat brain. Male Wistar rats were injected subcutaneously (s.c.) with 0.1, 0.25, or 1.0 mg/kg of haloperidol. Two h after the injection, the rats were perfused, and the numbers of Fos immunoreactive neurons were counted in 24 brain regions. In contrast to the limited changes in Fos immunoreactivity at the low dose of haloperidol (0.1 mg/kg), the moderate dose (0.25 mg/kg) induced widespread increases in Fos-positive neurons in the rat brain. Large increases were produced in the caudate-putamen, nucleus accumbens, central amygdaloid nucleus, dorsomedial hypothalamic nucleus, hippocampus CA1 and substantia nigra pars compacta. Moderate increases were observed in the entorhinal cortex, lateral septum, lateral habenula, lateral amygdaloid nucleus, dentate gyrus, and mesencephalic central grey. Mild increases were induced in the anterior cingulate, temporal, occipital and perirhinal cortex, and central medial thalamic nucleus. The distribution of changes in Fos immunoreactivity at the high dose of haloperidol (1.0 mg/kg) were comparable to their distribution at the moderate dose. These findings indicate that the effect of acute haloperidol on Fos expression is widely distributed in the rat brain beyond the previously known dopamine-rich areas at the dose which produces plasma levels equivalent to those within the therapeutic range used clinically in humans. Further studies on the effects of chronic antipsychotic treatment are needed in order to identify the sites of the therapeutic action of antipsychotic drugs.     

Effects of chronic treatment of olanzapine and haloperidol on peptide YY binding densities in the rat brain

This study examined regional changes of peptide YY (PYY) binding densities in the rat brain after chronic administration of olanzapine (1.2 mg/kg/day) and haloperidol (2.0 mg/kg/day) for 36 days. PYY binding densities and distributions were detected by [(125)I] binding autoradiography after rats were sacrificed either 2 h or 48 h after the last drug administration to examine both immediate and delayed effects following the drug withdrawal. Following 2 h of drug administration, it showed that olanzapine administration significantly decreased PYY binding densities, predominantly in the posterodorsal part of medial amygdaloid nucleus (52%, p<0.05), dorsal part of medial geniculate nucleus (56%, p<0.05), superficial gray layer of the superior colliculus (53%, p<0.05), parabrachial pigmented nucleus (54%, p<0.05) and periaqueductal gray (50%, p<0.05) compared to controls, while rebound increases of PYY binding densities were observed in most of examined regions 48 h later with only medial geniculate nucleus dorsal showing significant increase compared to controls (118%, p<0.01). In contrast, no change in PYY binding densities was found in the haloperidol-treated groups 2 h after drug administration, while only a significant rebound increase was observed in the dorsal part of medial geniculate nucleus (94%, p<0.01) 48 h after the haloperidol withdrawal. Alterations in PYY binding densities in brain regions such as the dorsal part of medial geniculate nucleus, superficial gray layer of superior colliculus, periaqueductal gray and parabrachial pigmented nucleus may represent the specific regions that mediate the clinical effects of antipsychotics via neuropeptide Y system. Our study also implicates NPY receptors as a novel therapeutic target in the treatment of psychotic disorders.     

两种方法提取脑组织总蛋白的色谱串联质谱分析

目的通过对提取大鼠脑组织总蛋白不同方法的研究,建立提取脑组织总蛋白的标准方法。方法在本实验中分别用总蛋白提取试剂盒和自制裂解液提取脑组织总蛋白,测量蛋白浓度。用体积排阻色谱（SEC）分离后分别进行基质辅助激光电离飞行时间质谱（MALDI-TOF-MS）做蛋白全谱。结果总蛋白提取试剂盒和自制裂解液总蛋白提取量分别为：（5.6±0.2）mg、（6.7±0.5）mg,MALDI-TOF-MS蛋白全谱的谱峰数分别为（304.2±22.3）和（331.6±30.5）个。结论自制裂解液与常规的总蛋白提取试剂盒相比具有蛋白提取率高、蛋白溶解性好等优点。     

Failure of chronic haloperidol treatment to alter levels of cholecystokinin in the rat brain striatum and olfactory tubercle-nucleus accumbens area

We have studied the effect of chronic haloperidol (HAL) treatment on CCK-8 levels in two rat brain regions. HAL administration using two different protocols, daily injections and infusion with subcutaneously implanted minipumps, did not produce any significant changes in CCK-8 levels in the striatum or olfactory tubercle-nucleus accumbens area.     

Greater resistance and lower contribution of free radicals to hypoxic neurotoxicity in immature rat brain compared to adult brain as revealed by dynamic changes in glucose metabolism

Seven-day-old rat brain slices were incubated at 36C in oxygenated Krebs-Ringer solution containing [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG), and serial two-dimensional time-resolved images of [(18)F]FDG uptake by the slices were obtained. The Gjedde-Patlak graphical method was applied to the image data, and the duration limit of hypoxia loading that allowed recovery of the fractional rate constant (k3*) of [(18)F]FDG (proportional to the cerebral glucose metabolic rate) after hypoxia loading to the unloaded control level was 50 min, and MK-801 as an N-methyl-D-aspartate antagonist had neuroprotective effects, but PBN as a free radical scavenger was ineffective. In our previous study in adult (7-week-old) rat brains [Murata et al., Exp Neurol 2000, 164:269-279], the limit of the hypoxia loading time was 20 min, and both MK-801 and PBN were effective. In the immature rat brains, the ratio of aerobic glucose metabolism to the total glucose metabolism was low compared with the adult rat brains, suggesting only a slight involvement of free radicals in hypoxic neurotoxicity. These data suggest that the higher resistance of immature brains to hypoxia compared to that of adult brains is attributable to a lower involvement of free radicals due to a lower aerobic glucose metabolic rate.     

SELDI-TOF-MS和双向凝胶电泳技术联用分析脑损伤后海马组织蛋白质表达谱的变化

目的 分析大鼠闭合性脑损伤后海马中蛋白质表达的变化及特点。 方法 选成年雄性SD大鼠随机分为手术对照组及损伤后不同时间点组, 复制Marmarou 落体打击闭合性颅脑损伤模型,应用蛋白质芯片技术和双向凝胶电泳技术分析大鼠脑损伤后海马中蛋白质表达的改变。并选择2个背景清晰、重复性及分辨率较好、在各组蛋白表达差异明显并呈现时序性变化的点进行质谱分析。结果 (1)蛋白质芯片结果显示,WCX-2芯片在海马中共获得364个蛋白质峰;IMAC-Cu芯片共获得345个蛋白质峰。 (2) 经双向凝胶电泳分离共获得18帧图,图谱蛋白质点分离效果良好,图谱平均匹配率＞80%。PDQuest软件对蛋白质图谱进行定量分析证实,相对分子质量为58.0×103的差异蛋白质为葡萄糖调节蛋白,在损伤后4h、8h和12h表达水平上调;相对分子质量为28.4×103的差异蛋白质为蛋白酶体α亚单位3型,损伤后4h、8h、12h、24h和48h表达水平均上调。 结论 脑损伤可引起海马中蛋白质表达谱发生变化。葡萄糖调节蛋白和蛋白酶体α亚单位3型可能与创伤后脑损伤的发生、发展密切相关。     

Small-scale purification and mass spectrometry analysis reveal a third aquaporin-4 protein isoform of 36kDa in rat brain

Aquaporin-4 (AQP4) is known to have two main isoforms M1 and M23 in the brain. Immunoblot analyses have provided evidence of additional AQP4 immunopositive bands, suggesting that the repertoire of AQP4 isoforms is broader than previously assumed. As isoforms beyond M1 and M23 are not observed in recombinant systems, investigation of novel isoforms requires the use of a native source. Here we report purification of AQP4 to three silver-stained proteins on SDS-PAGE. This was achieved by organelle separation, alkaline stripping of cellular membranes, detergent solubilization and multiple chromatographic steps. The three proteins that co-purified were identified as AQP4 by mass spectrometry. These results represent the first purification of AQP4 from a native source and demonstrate by mass spectrometry the presence of a third AQP4 isoform of 36kDa in the rat brain. Immunoblots revealed that the same isoform is present in the mouse, pig, and human brain.     

Neonatal phencyclidine treatment selectively attenuates mesolimbic dopamine function in adult rats as revealed by methamphetamine-induced behavior and c-fos mRNA expression in the brain

One of the major hypotheses regarding the pathogenesis of schizophrenia is the implication of neurodevelopmental abnormality. However, the mechanism of delayed onset of schizophrenic symptoms, in which increased dopaminergic activity in mesolimbic or mesocortical dopamine systems plays a pathological role, is not known. In this study, we investigated whether the chronic blockade of N-methyl-D-aspartate (NMDA) receptor by phencyclidine (PCP), an NMDA channel blocker, during development could disrupt the dopamine system during later life. Neonatal rats were injected with PCP subcutaneously daily from postnatal day (PD) 1 to PD 14 and their dopaminergic function was evaluated on PD 42 by rating the methamphetamine (MAP)-induced behavior. To illustrate the activated brain regions, the expression of c-fos mRNA in response to a MAP challenge was also studied utilizing in situ hybridization. Chronic neonatal PCP treatment attenuated MAP-induced oral stereotypy (licking and gnawing) and reduced MAP-induced expression of c-fos mRNA in the N. accumbens shell region and VTA but not in the N. accumbens core region, medial striatum, or substantia nigra. These results suggest that neonatal blockade of NMDA receptor, which induces a number of effects in the developing nervous system, may cause long-lasting functional changes of the mesolimbic dopamine system.     

Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-β-methylcholine (IS) from endogenous compounds and Ringer's salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringer's solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.     

The effect of chronic treatment with a novel aryl-piperazine antipsychotic on monoamine receptors in rat brain

The effects of chronic treatment of rats with RWJ 3776, a novel aryl-piperazine containing antipsychotic drug, on brain monoamine receptors were studied. Rats were treated daily with RWJ 37796 (1.3 mg/kg), the typical antipsychotic haloperidol (1 mg/kg) or vehicle (control) for 21 days, and were sacrificed 3 days after the last injection. Binding of [³H]Sch-23390 and [³H]spiperone to D₁ and D₂ dopamine receptors, respectively, and [³H]8-hydroxy-2-(di-n-propylamino)-tetralin ([³H]8OH-DPAT) to 5-HT_1A receptors were measured in various brain regions using quantitative autoradiography. Binding to D₂ dopamine receptors was significantly elevated in the caudate-putamen of rats treated with haloperidol or RWJ 37796 as compared to controls. However, the magnitude of the increase in D₂ binding was significantly greater in haloperidol-treated (+ 38%) compared to RWJ 37796-treated (+ 21%) rats. Haloperidol treatment also increased binding (+ 35%) to D₂ dopamine receptors in the nucleus accumbens, where RWJ 37796 treatment had a considerably smaller effect (+ 12). No changes in D₁ dopamine or 5-HT_1A receptor binding were detected following either antipsychotic treatment in any brain regions studied. Thus, at comparable doses, the novel antipsychotic RWJ 37796 produces less up-regulation of D₂ dopamine receptor binding in the striatum than does the typical antipsychotic haloperidol.     

Influence of acute, subchronic and chronic treatment with neuroleptic (haloperidol) on enkephalins and their precursors in the striatum of rat brain

We examined the effects of chronic, subchronic and acute treatment with haloperidol on the ME, the MERGL and enkephalin precursor concentrations in rat brain. The changes affected primarily the striatum. The ME content was greatly increased by the treatment, the precursor level was decreased by the haloperidol treatment. The specific mRNA for proenkephalin A increased. For these reasons, we conclude that the effect of haloperidol increase both the biosynthesis and the processing of precursors of enkephalins in the striatum.     

Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.

An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.