Norma M. Giusto

Age-Associated Changes in Central Nervous System Glycerolipid Composition and Metabolism*

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.

Age-associated changes in the content and fatty acid composition of brain glycerophospholipids.

The fatty acid composition of total lipids and individual phospholipids and the ratio of cholesterol-to-phospholipid content were studied in cerebral cortex, subcortical white matter, cerebellum and medulla oblongata/pons in 4-, 21.5- and 28-month-old rats. The cholesterol-to-phospholipid molar ratio in subcortical white matter, medulla oblongata/pons and cerebellum in 28-month-old rats was found to be 17, 17 and 16% higher, respectively, than in adult rats. These alterations in the molar ratio were produced as a result of a net increase in cholesterol content rather than by changes in the total phospholipid content. The content of alkenylacylglycerophosphoethanolamine (alkenylacyl GPE) increased in 28-month-old rats with respect to 4-month-old rats, following the order cerebral cortex > cerebellum > medulla oblongata/pons > subcortical white matter. The fatty acid composition of total lipids showed an increase in monounsaturated fatty acids (MUFA) and a decrease in polyunsaturated fatty acid (PUFA) content with increasing age in all regions studied, such changes being more marked in cerebellum, medulla oblongata/pons and subcortical white matter than in cerebral cortex. The proportion of 22:6(n-3) in cholineglycerophospholipids in the different brain regions of 28-month-old rats showed a slight decrease with respect to that in adult rats following the order cerebellum > medulla oblongata/pons > subcortical white matter, whereas that of 20:4(n-6) decreased only in cerebellum. Ethanolamineglycerophospholipid fatty acid composition was modified in 28-month-old rats through a marked increase in monounsaturated fatty acids (18:1(n-9) and 20:1(n-9) specifically) in all brain areas. The MUFA content of alkenylacyl GPE increased with increasing age in all the regions studied, in the order cerebral cortex > medulla oblongata/pons > subcortical white matter > cerebellum. An increase in the MUFA content of diacylglycerophosphoethanolamine was observed in medulla oblongata/pons and cerebellum of aged rats with respect to control rats. PUFA content of alkenylacyl GPE decreased mainly in medulla oblongata/pons, cerebral cortex and subcortical white matter of aged rats. The PUFA content of serineglycerophospholipids was the most affected by aging. Changes occurred mainly in the content of 22:6(n-3), 22:4(n-6) and 20:4(n-6) in cerebellum and of 22:6(n-3) and 22:4(n-6) in subcortical white matter. Changes in the inositolglycerophospholipid fatty acid content of 28-month-old rats were observed mainly in medulla oblongata/pons, which showed a decrease in PUFA (22:4(n-6) and 22:6(n-3)) content and an increase in MUFA (18:1 and 20:1).

Iron and Mechanisms of Neurotoxicity

The accumulation of transition metals (e.g., copper, zinc, and iron) and the dysregulation of their metabolism are a hallmark in the pathogenesis of several neurodegenerative diseases. This paper will be focused on the mechanism of neurotoxicity mediated by iron. This metal progressively accumulates in the brain both during normal aging and neurodegenerative processes. High iron concentrations in the brain have been consistently observed in Alzheimers (AD) and Parkinsons (PD) diseases. In this connection, metalloneurobiology has become extremely important in establishing the role of iron in the onset and progression of neurodegenerative diseases. Neurons have developed several protective mechanisms against oxidative stress, among them, the activation of cellular signaling pathways. The final response will depend on the identity, intensity, and persistence of the oxidative insult. The characterization of the mechanisms mediating the effects of iron-induced increase in neuronal dysfunction and death is central to understanding the pathology of a number of neurodegenerative disorders.

Effects of aging on the content, composition and synthesis of sphingomyelin in the central nervous system

Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.5, 21.5 and 26.5-month-old rats. SPH content increased in the cerebral hemispheres, cerebellum and medulla oblongata plus pons as age increased. The highest SPH content was observed in 26.5-month-old rats, with values increasing by 1.74, 2.75 and 0.88-fold, respectively, over 2.5-month-old rats. The SPH fatty acid composition of brains from aged rats was markedly different from that of adult rats. Between 2.5 and 26.5 months of age the monoenoic/saturated fatty acid ratio increased from 0.22, 0.30 and 0.54 to 0.54, 0.68 and 1.03 in cerebral hemispheres, cerebellum and medulla oblongata plus pons, respectively. The percentage and content of fatty acids longer than 22 carbon atoms esterified to SPH increased with age from 18, 26 and 44 to 48, 52 and 62 mole % in cerebral hemispheres, cerebellum and medulla oblongata plus pons in 26.5-month-old rats. In subcortical white matter from aged rats, monoenoic 22–26 carbon atom fatty acids increased more than the saturated ones in 21.5-month-old rats relative to 2.5-month-old rats.In vitro synthesis of SPH from [3H]choline and [3H]palmitic acid in cerebral cortex and cerebellum showed no significant differences between adult rats and those 21.5 months of age. In cerebellum and in cerebral cortex, [14C] serine incorporation increased in aged rats. The results suggest that aging induces increases in both SPH content and in the monoenoic/saturated fatty acid ratio. These increases are quantitatively different in all brain regions analyzed.

Insulin-like growth factor-I is a potential trophic factor for amacrine cells

In this study we show that insulin‐like growth factor (IGF)‐I selectively promotes survival and differentiation of amacrine neurons. In cultures lacking this factor, an initial degeneration pathway, selectively affecting amacrine neurons, led to no lamellipodia development and little axon outgrowth. Cell lysis initially affected 50% of amacrine neurons; those remaining underwent apoptosis leading to the death of approximately 95% of them by day 10. Apoptosis was preceded by a marked increase in c‐Jun expression. Addition of IGF‐I or high concentrations (over 1 µm) of either insulin or IGF‐II to the cultures prevented the degeneration of amacrine neurons, stimulated their neurite outgrowth, increased phospho‐Akt expression and decreased c‐Jun expression. The high insulin and IGF‐II concentrations required to protect amacrine cells suggest that these neurons depend on IGF‐I for their survival, IGF‐II and insulin probably acting through IGF‐I receptors to mimic IGF‐I effects. Inhibition of phosphatidylinositol‐3 kinase (PI 3‐kinase) with wortmannin blocked insulin‐mediated survival. Wortmannin addition had similar effects to IGF‐I deprivation: it prevented neurite outgrowth, increased c‐Jun expression and induced apoptosis. These results suggest that IGF‐I is essential for the survival and differentiation of amacrine neurons, and activation of PI 3‐kinase is involved in the intracellular signaling pathways mediating these effects.

Effects of aging on the composition and metabolism of docosahexaenoate-containing lipids of retina

The amount of docosahexaenoate (22∶6n−3)-containing phospholipid species decreases with aging in the rat retina. Most lipids, but especially choline and serine glycerophospholipids, show a significant fall in 22∶6n−3, which is not compensated by increases in other polyenoic fatty acids. The decrease not only affects 22∶6 but also various very long chain n−3 hexaenoic fatty acids which, in phosphatidylcholine, have up to 36 carbon atoms, and which are probably synthesized by successive elongations of 22∶6n−3. The in vitro incorporation of [2-3H] glycerol into retinal lipids indicates that the de novo biosynthetic pathways are not impaired by aging. The incorporation of [1-14C]docosahexaenoate is significantly stimulated into all lipids of aged retinas, but to the largest extent in those showing the largest decreases in 22∶6, especially in choline glycerophospholipids. The results indicate that the decreased levels of 22∶6 with aging are due not to an impaired activity of the enzymes involved in the synthesis and turnover of phospholipids but to a decreased availability of this polyene in the retina. It is suggested that this may stem from a defect in some of the enzymatic steps that lead to the synthesis of 22∶6n−3, probably that catalyzed by Δ4 desaturase, the effect on longer hexaenes being secondary to the decreased synthesis of 22∶6.

High content of 22:6 (docosahexaenoate) and active [2-3h]glycerol metabolism of phosphatidic acid from photoreceptor membranes

This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phosphatidic acid of rod outer segment membranes from vertebrate retinas. A relatively high content of phosphatidic acid was observed in rod outer segment membranes isolated from rat, toad and bovine retinas. In bovine retinas, about 65% of the acyl groups of phosphatidic acid were composed of docosahexaenoate. Arachidonate and docosapentaenoate represented about 4 and 5%, respectively, of the total, whereas stearate was the most common saturated acyl chain. An active [2-3H]glycerol metabolism in the phosphatidic acid of these membranes was found when whole retinas were incubated with the precursor for short periods prior to subcellular fractionation. Our results suggested that the pool of phosphatidic acid enriched in docosahexaenoate may arise from de novo biosynthesis or from phospholipid degradation by a phospholipase D enzyme, and that it is not metabolically related, in any major fashion, to the diacylglycerols of rod outer segment membranes.

Phospholipids and acylglycerols biosynthesis and 14CO2 production from [14C]glycerol in the bovine retina: The effects of incubation time, oxygen and glucose

Abstract When the entire bovine retina was incubated in 95 μ m [U-14C]glycerol, the precursor penetrated the tissue and it was used for the de novo biosynthesis of glycerolipids. A minor proportion of the labeled glycerol turned to 14CO2. The following labeling sequence was suggested: glycerol phosphorylation, double acylation, phosphatidate, diacylglycerol and triacylglycerol. Under aerobic conditions, a remarkable channelling of the label towards triacylglycerol biosynthesis was found. Comparatively, much lower specific activities were encountered in all the phosphoglycerides, phosphatidylinositol biosynthesis attaining the highest rate. At an early incubation time, the peak labeling of total phosphoglycerides was closely followed by phosphatidate radioactivity. The phosphoglyceride content slightly decreased in retinas incubated in the absence of added glucose. Anoxia, however, resulted in a diminution of the concentration of the diacylglycerophosphatides of choline and of ethanolamine and in a significant increase of their monoacyl derivatives. In an incubation medium devoid of glucose, the specific activity in the diacyl and monoacylglycerophosphatides of choline and ethanolamine was significantly raised. Furthermore, there was a dramatic decrease in the triacylglycerol biosynthesis without modification of glycerol incorporation in the polar lipids and augmented the labeling in the lipid-free tissue residue. Anoxia abolishes the uptake of glycerol in retina lipids.

Phosphatidic acid of retinal microsomes contains a high proportion of docosahexaenoate.

Abstract Microsomes obtained from the bovine retina contain phosphatidic acid enriched in polyenoic fatty acids. A relatively high proportion of docosahexaenoate, not reported previously in phosphatidic acid of any subcellular fraction, is the main feature of this phospholipid. It is suggested that acylation by docosahexaenoyl-CoA takes place prior to the synthesis of phosphatidic acid in retinal microsomes.

CHARACTERIZATION OF PHOSPHOLIPASE D ACTIVITY IN BOVINE PHOTORECEPTOR MEMBRANES

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[23H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol × h−1 × (mg protein)−1. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol × h−1 × (mg protein)−1. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.