Thomas Fraser

Fatty Acid Composition of Frontal, Temporal and Parietal Neocortex in the Normal Human Brain and in Alzheimer’s Disease

Dietary ω3-polyunsaturated fatty acids are thought to influence the risk of Alzheimer’s disease (AD), and supplemental docosahexaenoic acid (DHA; 22:6n-3) has been reported to reduce neurodegeneration in mouse models of AD. We have analysed the fatty acid composition of frontal, temporal and parietal neocortex in 58 normal and 114 AD brains. Significant reductions were found for stearic acid (18:0) in frontal and temporal cortex and arachidonic acid (20:4n-6) in temporal cortex in AD, and increases in oleic acid in frontal and temporal cortex (18:1n-9) and palmitic acid (16:0) in parietal cortex. DHA level varied more in AD than controls but the mean values were not significantly different. Fatty acid composition was not related to APOE genotype, age, gender or post-mortem delay. Further research is needed to distinguish between alterations that are secondary to AD and those that contribute to the disease process.

Oxidative Balance in Alzheimer's Disease: Relationship to APOE, Braak Tangle Stage, and the Concentrations of Soluble and Insoluble Amyloid-β

Oxidative damage is greater in brain tissue from patients with Alzheimers disease (AD) than age-matched controls. The timing of this damage in relation to other pathogenic processes in AD remains unclear. We have examined the relationship of lipid peroxidation (thiobarbituric acid-reactive substances; TBARS) and antioxidant capacity (Trolox-equivalent) to APOE status, Braak tangle stage, amyloid-β (Aβ) plaque load, and the concentration of soluble and insoluble forms of Aβ, post-synaptic and dendritic spine proteins PSD95 and drebrin, β-secretase and Aβ-degrading enzymes neprilysin (NEP), insulin-degrading enzyme (IDE), and angiotensin-converting enzyme (ACE), in frontal, temporal, and parietal cortex from AD and control brains. Antioxidant capacity was significantly elevated in AD and directly related to disease severity as indicated by Braak tangle stage and the amount of insoluble Aβ. APOE ε4 was associated with increased antioxidant capacity in AD but not controls. In contrast, apart from a reduction in TBARS in Braak stages III-IV in frontal cortex, this measure of oxidative damage did not change significantly with any indicator of disease severity. It was, however, higher in APOE ε4-positive than ε4-negative AD patients and correlated with β-secretase activity. Neither antioxidant capacity nor oxidative damage was related to the level of PSD95 or drebin or the activity of NEP, IDE, or ACE. Antioxidant capacity in AD is closely related to the level of insoluble Aβ and increases with pathological progression of the disease. Increased β-secretase activity associated with oxidative stress is likely to contribute to the accumulation of Aβ and this, in turn, to induce antioxidant capacity.

The variant ‘his‐box’ of the C18‐Δ9‐PUFA‐specific elongase IgASE1 from Isochrysis galbana is essential for optimum enzyme activity

IgASE1, a C18‐Δ9‐polyunsaturated fatty acid‐specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his‐box) with glutamine replacing the first histidine of the conserved histidine‐rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his‐box of IgASE1 was determined by site‐directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate specificity was shown to be unaffected by these changes suggesting that components of the his‐box are not directly responsible for substrate specificity.

Low-temperature improved-throughput method for analysis of brain fatty acids and assessment of their post-mortem stability

Deficiency of docosahexaenoic acid (DHA) and other omega-3 (omega3) fatty acids may constitute an alterable risk factor for Alzheimers disease (AD). Mechanisms of potential involvement of DHA in the disease process have been postulated primarily from studies in vitro and in mouse models of AD. Information on the fatty acid profile of the brain in AD itself is limited and in some respects contradictory. Interpretation of the findings is complicated by the diversity of methods used in previous studies and a lack of information as to the effect of post-mortem delay on the results. Here we report the development of a simple and highly reproducible method that enables relatively high-throughput measurement of the fatty acid composition in samples of brain tissue and using this method we have demonstrated that there is no significant change in fatty acid composition under conditions designed to model post-mortem delay of up to 3 days at 4 degrees C (or even at room temperature). The development of this method and the observation that delay of up to 3 days has no effect on fatty acid content will facilitate further studies of fatty acid composition on large cohorts of post-mortem brains.

Does triacylglycerol biosynthesis require diacylglycerol acyltransferase (DAGAT)

Microsomal membrane preparations from the developing seeds of sunflower (Helianthus annuus L.) catalyse the conversion of sn-glycerol-3-phosphate and acyl-CoA to triacylglycerol via phosphatidic acid and diacylglycerol. The formation of diacylglycerol from phosphatidic acid was Mg2+ dependent and in the presence of EDTA phosphatidic acid accumulated. This property was used to generate large quantities of endogenous radioactive phosphatidic acid in the membranes. On addition of Mg2+ the phosphatidic acid was used in triacylglycerol formation. Acyl-CoA had little effect on the label which accumulated in triacylglycerol from phosphatidic acid. Diacylglycerol acyltransferase, therefore, may not play a major role in oil formation as originally envisaged and other enzymes, including diacylglycerol:diacylglycerol transacylase [Stobart, Mancha, Lenman, Dahlqvist and Stymne (1997) Planta 203, 58-66] may have important biosynthetic functions.

Kinetic Analysis of Microsomal SN -Glycerol-3-Phosphate Acyltransferase from the Developing Seeds of Sunflower and Mortierella Alpina

Glycerol-3-phosphate acyltransferase (GPAT) is the first committed step in glycerolipid synthesis and presumed to be rate-limiting. It catalyses the esterification of the sn-1 hydroxyl of glycerol-3-phosphate (G3P) with an activated fatty acid forming 1-acyl-sn-glycerol-3-phosphate (LPA). Kinetic data reported here are consistent with the GPAT catalysed acylation of G3P occurring by single and double displacement reaction mechanisms in sunflower and Mortierella alpina, respectively, and therefore suggest that there may be no evolutionary link between fungal and higher plant microsomal GPAT.

Membrane-Bound SN -2-Monoacylglycerol Acyltransferase (MGAT) is Involved in Triacylglycerol Synthesis

Sunflower microsomal preparations catalysed the incorporation of radioactivity from [14C]oleoyl-CoA in the presence of sn-2-monoacylglycerol (2-MAG) Similar results were obtained using sn-2-mono-[14C]oleoylglycerol and non-radioactive acyl-CoA. The observations were consistent with the activity of a monoacylglycerol acyltransferase (MGAT, E.C. 2.3.1.22.) catalysing the acylation of MAG yielding diacylglycerol (DAG), and the rapid utilization of DAG in TAG synthesis. The MGAT was particularly efficient with the 2-MAG substrate and was selective for monounsaturated acyl species. Similar membrane-bound MGAT was also present in linseed (Linum usitatissimum L.), Mortiella alpina, Candida curvata D. and Saccharomyces cerevisiae.

Oil Biosynthesis in Mortierella Alpina Microsomal Membranes

Microsomal membrane preparations from Mortierella alpina incubated with sn-glycerol-3-phosphate (G3P) and [14C]oleoyl-CoA catalysed the formation of radioactive phosphatidic acid (PA), diacylglycerol (DAG), triacylglycerol (TAG) and phosphatidylcholine (PC). A similar pattern of radiolabelling was observed when [14C]G3P and non-labelled oleoyl-CoA were used except that no radioactivity was found in PC. This lipid was, however, labelled from [14C]G3P and oleoyl-CoA in the presence of CDP-choline. Equilibration between [14C]glycerol labelled DAG and PC in M. alpina microsomal membranes was dependent on the addition of cytidine diphosphocholine (CDP-choline) A concomitant decrease in label in DAG and TAG occurred in conjunction with the increase in radioactivity in PC. The results indicate that PC and TAG synthesis via diacylglycerol:cholinephosphotransferase (DAG:CPT) and diacylglycerol acyltransferase (DAGAT), respectively, share a common pool of DAG.

Fungal Oil Biosynthesis

γ-linolenic acid (GLA; 18:3 Δ6,9,12) is produced by only a few higher plant species and is also found in the oil of some fungi. Studies on the fungus Mucor circinelloides indicated that the major site for Δ6 desaturation was position sn-2 of PI [1]. It also has been proposed that in the fungus Mucor INMI Δ6 desaturation can occur on PC, PE and acyl-CoA [2]. We have studied fatty acid synthesis and oil formation in Mucor circinelloides, a fungus which accumulates oil rich in polyunsaturated fatty acids.