Membrane Lipids in the Aging Human Brain

The membrane lipid composition of human brain has been studied in 21 men and 18 women 60-97 years of age. This brain tissue series is unique, because it has been obtained only from individuals who lived a normal social life in their own homes and died suddenly and unexpectedly from arteriosclerotic heart disease or ruptured aortic aneurysms. They had no history of neurological or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The percentage of solids diminished continuously during the whole period, but the marked individual differences suggested large variations in the hydration of the brain. The content of membrane lipids also diminished continuously up to 90 years of age, when a marked diminution in level of gangliosides and cerebrosides occurred, a result indicating a rapid reduction in amount of neuronal membranes and myelin. The clinical implications of the variation in brain hydration and the rapid loss of membrane lipids after 90 years of age are discussed.     

Gangliosides and Other Lipids of the Growth Cone Membrane

Growth cone membranes, derived from growth cone particles isolated from 16- to 18-day-old fetal rat brain, were found to be rich in overall lipid content with a lipid-to-protein ratio of 3.5. The phospholipid-to-cholesterol ratio indicated considerably less cholesterol than plasma membranes from mature neurons. All major classes of phospholipid were present in the usual proportions except sphingomyelin, which could not be detected. Gangliosides expressed in relation to protein were present at somewhat higher levels compared to previously reported values for synaptic plasma membranes (73 versus 44 micrograms/mg protein), but when related to phospholipid their level was well below that of the latter (26 versus 62 micrograms/mg phospholipid). The ganglioside pattern was generally similar to that of mature synaptic membranes except for the presence of relatively more GD3 and less GD1a, a phenomenon also observed in whole fetal brain of the same age. Several neutral glycosphingolipids were detected, glucosylceramide being the major one of this group. Their total level in growth cone membranes was roughly comparable to that of gangliosides, but unlike the latter their concentration in whole brain decreased with development. For comparison we analyzed the ganglioside composition of mixed membrane fractions from the same fetal brains and found no significant differences between these and growth cone membranes, suggesting that these glycoconjugates are not localized specifically in the growth cones. Neutral glycosphingolipids, on the other hand, appeared somewhat more concentrated in growth cones than in the mixed membranes.     

Phospholipid and Ganglioside Composition in Rat Brain After Chronic Intake of Ethanol

Abstract: A total of 18 60-day-old male Wistar rats were divided into three groups of six animals each. One group was fed a basal diet containing high levels of protein, fat, carbohydrate, vitamins, and minerals and separately a solution of 25% sucrose-32% ethyl alcohol (wt/vol). A second group was offered water as the only drinking fluid and a similar solid diet, except that carbohydrate replaced ethanol isocalorically. A third group was maintained on the basal diet ad libitum . All groups of animals were killed in a sober state after 6 months of chronic ethanol treatment and lipid analyses were performed on brain homog-enates. Chronic treatment of the animals with ethanol produces statistically significant modification of the phospholipid and ganglioside patterns in rat brain. A statistically significant decrease of the total phospholipid content and of some of the investigated fractions, i.e., phos-phatidylcholine and phosphatidylserine, as well as an increase of phosphatidylinositol were observed. Chronic alcohol consumption was associated with a statistically significant increase in the total amount of ganglioside in rat brain. An increase in most of the investigated ganglioside fractions was indicated but the difference was statistically significant only for trisialoganglioside GT_1b. The amount of disialoganglioside GD_1a in these brains was decreased after chronic intake of ethanol.     

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12-18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two- to threefold as cell density increased 25-fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di- and trisialogangliosides (GD₂+ GD_lb+ GT₁) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM₄+ GM₃+ GM₂) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM₄+ GM₃+ GM₂) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM₃ (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GM_t (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.     

Lipid Composition in Different Regions of the Brain in Alzheimer''s Disease/Senile Dementia of Alzheimer''s Type

The lipid compositions of 10 different brain regions from patients affected by Alzheimer's disease/senile dementia of Alzheimer's type were analyzed. The total phospholipid amount decreased somewhat in nucleus caudatus and in white matter. The cortical areas that are morphologically affected by Alzheimer's disease, i.e., frontal and temporal cortex and the hippocampus, showed elevated contents of lipid solvent-extractable phosphatidylinositol. Sphingomyelin content was decreased in regions rich in myelin. There was a 20-50% decrease in dolichol amount in all investigated parts of the brain, but no change was seen in the polyisoprenoid pattern. Levels of alpha-unsaturated polyprenes were decreased in Alzheimer brains. Dolichyl-phosphate content increased in most regions, up to 100%. In both control and Alzheimer tissue almost all of the dolichyl-phosphate was covalently bound, apparently through glycosylation. Cholesterol amounts were highly variable but mostly unchanged, whereas ubiquinone concentrations increased by 30-100% in most regions in brains affected by Alzheimer's disease. These results demonstrate that both phospholipids and neutral lipids are modified in brains affected by Alzheimer's disease/senile dementia of Alzheimer's type.     

Distribution of Dolichol and Dolichyl Phosphate in Human Brain

Autopsy material from deceased individuals between ages 2 and 90 was used to prepare cerebellum, pons, and other selected regions of the brain, the spinal cord, and peripheral nerves. The concentration of dolichol in these different tissues varied greatly and the increase in concentration during the life span varied between 2.5- and 21-fold. In contrast, dolichyl-phosphate (dolichyl-P) was more evenly distributed in these tissues and its concentration increased to a moderate extent only during childhood. The level of cholesterol displayed smaller regional differences and decreased about 15% between ages 35 and 90. Differences in the total phospholipid content were limited. These results demonstrate enrichment and individual regulation of various lipids in specialized regions of the human brain. The independent regulation of dolichol and dolichyl-P levels in the brain and the possible role of dolichol in the function of the aging nerve cell are also emphasized.     

Effects of Nutritional Rehabilitation on the Content and Lipid Composition of Brain Gray and White Matter of Neonatally Undernourished Rats

Abstract: Neonatal undernutrition affects the content and lipid concentrations of gray and white matter. Nutritional rehabilitation reverses the deficit observed in gray matter. In the case of white matter the lipid concentration but not the content comes back to normal.     

Cholesterol Esters of the Human Brain During Fetal and Early Postnatal Development: Content and Fatty Acid Composition

Abstract: The content and fatty acid composition of cholesterol esters of the human brain during development from 13 weeks' gestation up to 26 months of age was studied. The three major brain areas, the forebrain, cerebellum, and the brain stem, were studied separately. The concentration of the esters in each brain region was the highest at the earliest fetal age of 13 weeks and fell during growth. However, transient rises in the concentration were observed, at about birth in the forebrain and at 4–5 months after birth in the cerebellum The peak concentration during the transient period (125–150 μg/g fresh tissue of forebrain and 100–125 μg/g of cerebellum) was similar to the concentrations observed in the two parts respectively during early fetal ages. The brain stem also showed similar transient peak at about a few weeks before birth, but only when the esters were expressed as amount per cell. In absolute terms, a clear transient period was evident in the forebrain between birth and 9 months, while in the cerebellum or the brain stem, the total amount of the esters increased up to about 1 year of age and then remained almost unchanged. The major fatty acids of the esters were palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acid. Most of these fatty acids showed certain changes in relative proportions during development. Thus, in the forebrain, palmitic and oleic acid decreased from about 32% and 40% (weight percentages) at 13–15 weeks of gestation to about 20% and 25% respectively at 26 months of age. During this period, linoleic and arachidonic acid increased from about 3% and S% to about 10% and 24%, respectively. Most of these changes occurred after birth. The cerebellum and the brain stem differed only slightly from the forebrain in either the fatty acid composition or the pattern of the developmental changes in the composition.     

Cholesterol Esters in Rat Brain Infected with Acute Measles Encephalitis: Concentration and Fatty Acid Composition

The present study deals with the concentration and fatty acid composition of cholesterol esters in rat brains infected experimentally with measles virus to induce acute encephalitis. The left side of the cerebrum, as well as other portions of the brain, when inoculated percutaneously contained a large amount of cholesterol esters. The major fatty acids from the esters in the brain were C16:0, C16:1, C18:0, and C18:1; those from the serum were C18:1, C18:2, and C20:4. This result indicates that cholesterol esters may not come from serum but can be synthesized in situ, even in the brain with acute viral infection.     

Detection of Cystathionine Ketimine and Lanthionine Ketimine in Human Brain

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.     

Regional Localization and Subcellular Compartmentalization of Thyrotropin-Releasing Hormone in Adult Human Brain

In the current study, we sought to define the subcellular compartmentalization of thyrotropin-releasing hormone (TRH) in adult human brain tissues. Upon evaluating tissues (3-24 h post mortem) from 62 humans, ranging in age from 5 to 75 years, we found that TRH was widely distributed throughout the brain. The highest TRH concentration (ng/mg protein) was in the stalk-median eminence region of the hypothalamus (19.3 +/- 3.3, mean +/- SE); the TRH concentration in the hypothalamus, exclusive of the stalk-median eminence, was much lower (1.7 +/- 0.2). Substantial quantities of TRH also were detected in the medulla oblongata (0.26 +/- 0.08), mammillary bodies (0.33 +/- 0.25), and optic chiasm (0.14 +/- 0.07). Lower levels of TRH were found in the amygdala (0.060 +/- 0.015) and the corpus striatum (0.033 +/- 0.010). TRH was near or below the limits of detection in tissues of the cerebral and cerebellar cortices, the olfactory bulbs, the pons, and the hippocampus. When homogenates of medial basal hypothalamic tissue (prepared in 0.32 M sucrose-10 microM CaCl2) were fractionated by means of differential centrifugation, most of the TRH was recovered in subcellular particles which were pelleted at 10,000 X g and which contained the highest amounts of occluded LDH activity. When the nuclei-free supernatant fluid (900 X g S) was fractionated on discontinuous sucrose density gradients or continuous sucrose density gradients, most of the TRH was recovered in subcellular fractions containing synaptosomes. The subcellular distribution of TRH appeared to be stable for up to 24 h post mortem in rat and human brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

植物适应长期缺钾——积累叶片膜脂、维持根膜脂组成不变

环境对植物的胁迫可能是短期快速的、也可能是长期而缓慢的,而植物应对这两种胁迫的策略可能不同。膜脂组成变化是植物响应环境胁迫的主要手段之一,其响应长期胁迫和短期胁迫的样式也可能不同。植物膜脂组成对短期缺钾胁迫的响应已经有报道,但是对长期缺钾的响应如何尚且未知。我们设置了4种（51,051,0051和0mmol·L-1）不同的钾浓度,比较了拟南芥（Arabidopsis thaliana）及其生长于贫钾生境中的近缘种须弥芥（Crucihimalaya himalaica）长期缺钾后（18天）的生理和生化变化,发现须弥芥具有耐受贫钾的能力。我们进一步运用脂类组学的方法检测比较了拟南芥和须弥芥在长期缺钾胁迫下脂类组成的变化,发现：(1) 两种植物叶片中总脂以及几乎所有脂类的含量明显上升;(2) 两种植物都是地上部分膜脂的变化幅度大于根部膜脂的变化幅度;(3) 地上部分膜脂变化幅度,须弥芥的大于拟南芥的;地下部分的膜脂变化幅度,须弥芥的小于拟南芥的;(4) 拟南芥叶片和根中PA的含量显著上升,与PA相对应的是PE含量的显著下降,由此我们推测拟南芥中PA的积累主要来自于PE的水解。上述结果提示,在细胞水平上,植物主要通过积累叶片膜脂和维持根部膜脂组成基本不变来适应长期缺钾。     

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale. There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation. However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons. Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms. Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation. We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons. This approach is likely to have broad utility in the field of neuronal endocytic trafficking.     

Expression of Bcl-2 in Adult Human Brain Regions with Special Reference to Neurodegenerative Disorders

Abstract: The expression of the protooncogene bcl-2 , an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl-2 protein was detected by western blot analysis and immunohistochemistry. Two bcl-2 mRNA species were identified in the human brain. The pattern of distribution of bcl-2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl-2 mRNA was detected in some but not all neurons. In the regions examined for Bcl-2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimer's and Parkinson's diseases, quantification of bcl-2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.     

Lipid Metabolism and Membrane Composition Are Altered in the Brains of Type II Diabetic Mice

Abstract: CBL/57 strain db/db mice exhibit type II (non-insulin-dependent) diabetes. The affected mice are markedly hyperinsulinemic, hyperglycemic, and hypercholesterolemic, and their serum K⁺ levels are decreased. The brains of the diabetic mice are significantly smaller than those of their lean, control littermates, but the protein concentration is normal. The low brain weight is accompanied by a loss of major fatty acid components within the whole brain, nerve endings, and mitochondrial membranes. Cholesterol levels are low in whole brain but are not significantly different from normal in the synaptosomal membranes. The phospholipid concentration is significantly decreased in whole brain homogenates, crude synaptosomal membranes, and crude mitochondrial membranes of the diabetic mice. In addition, the specific activities of membrane-bound synaptosomal acetylcholinesterase, Na⁺,K⁺-ATPase, and Mg²⁺-ATPase are decreased in crude synaptosomal membranes of the diabetic mice. The specific activities of carnitine palmitoyltransferase I and carnitine acetyltransferase are significantly increased in the crude mitochondrial fraction isolated from the brains of the type II diabetic mice, whereas the specific activity of pyruvate dehydrogenase complex is decreased. The specific activities of two other mitochondrial enzymes—monoamine oxidase B and citrate synthase—and a cytosolic enzyme—lactate dehydrogenase—are unaltered. The ability to synthesize cyclic AMP is markedly decreased in the brains of the diabetic mice. The concentrations of carnitine and of the amino acids, glutamate, aspartate, glutamine, and serine are unaltered, whereas glycine levels are significantly elevated in the brains of the db/db mice. The data suggest that in vivo the brains of the diabetic mice exhibit a decreased capacity for glucose oxidation and increased capacity for fatty acid oxidation. This hypothesis is supported by the finding that cerebral mitochondria isolated from the db/db mice oxidize [1-¹⁴C]palmitate to ¹⁴CO₂ at a rate almost twice that of control mitochondria. The present findings emphasize the potentially serious alteration of brain metabolism in uncontrolled type II diabetes.     

Concentration of Nucleosides and Related Compounds in Cerebral and Cerebellar Cortical Areas and White Matter of the Human Brain

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements.2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses.3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean±SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3±0.6, inosine 69.5±1.7, guanosine 13.5±0.4, uridine 52.4±1.2, uracil 8.4±0.3, hypoxanthine 108.6±2.0 and xanthine 54.8±1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent.4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.     

Mucopolysaccharidosis Types IH, IS, II and IIIA: Glycosaminoglycans and Lipids of Isolated Brain Cells and Other Fractions from Autopsied Tissues

Abstract: Brain cellular fractions were prepared in bulk from four non-neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8-month-old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one-half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four- to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides G_{m 2}, G_{m 3}, G_{d 3} and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of G_{m 3}, G_{m 2} and G_{d 3} together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.     

内质网应激条件下血管内皮细胞生长因子在人脑微血管内皮细胞中的表达

为观察内质网应激条件下血管内皮细胞生长因子的表达情况,用不同浓度的衣霉素处理体外培养的人脑微血管内皮细胞,建立内质网应激模型,采用RT—PCR、蛋白质免疫印迹以及免疫细胞化学的方法检测了细胞内血管内皮细胞生长因子的表达。结果发现血管内皮细胞生长因子在人脑微血管内皮细胞中存在一定的表达;内质网应激可诱导血管内皮细胞生长因子表达升高,随着衣霉素浓度的增高,血管内皮细胞生长因子的表达逐渐增加,与mRNA水平相比,血管内皮细胞生长因子蛋白量的增加更明显。实验结果提示人脑微血管内皮细胞中存在血管内皮细胞生长因子自分泌,血管内皮细胞生长因子可能是内质网应激的靶基因。     

Alternative Splicing,Expression and Cellular Localization of Calneuron-1 in the Rat and Human Brain

Calneuron-1 and -2 are members of the neuronal calcium-binding protein family (nCaBP). They are transmembrane Calmodulin-like EF-hand Ca²⁺-sensors, and a function in the control of Golgi-to-plasma membrane vesicle trafficking has been assigned to both proteins. In this paper, we describe the distribution of Calneuron-1 in rat and human brains. We show that Calneuron-1 is ubiquitously expressed in all brain regions examined. The protein is most abundant in Purkinje cells of the cerebellum and principal neurons of the cortex and limbic brain whereas no expression in glial cells is apparent. In addition, we identify two novel splice isoforms of Calneuron-1 with extended N-termini. These isoforms are particular abundant in the cerebellum. Taken together, these data set grounds for a better understanding of the cellular function of Calneurons.