Hannah Tayler

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.

Distribution and expression of picalm in Alzheimer disease.

PICALM, the gene encoding phosphatidylinositol-binding clathrin assembly (picalm) protein, was recently shown to be associated with risk of Alzheimer disease (AD). Picalm is a key component of clathrin-mediated endocytosis. It recruits clathrin and adaptor protein 2 (AP-2) to the plasma membrane and, along with, AP-2 recognizes target proteins. The attached clathrin triskelions cause membrane deformation around the target proteins enclosing them within clathrin-coated vesicles to be processed in lysosomes or endosomes. We examined the distribution of picalm in control and AD brain tissue and measured levels of picalm messenger RNA (mRNA) by real-time polymerase chain reaction. Immunolabeling of brain tissue showed that picalm is predominately present in endothelial cells. This was further supported by the demonstration of picalm in human cerebral microvascular cells grown in culture. Picalm mRNA was elevated in relation to glyceraldehyde-3-phosphate dehydrogenase but not factor VIII-related antigen or CD31 mRNA in the frontal cortex in AD. No change was seen in the temporal cortex or thalamus. The transport of A&bgr; across vessel walls and into the bloodstream is a major pathway of A&bgr; removal from the brain and picalm is ideally situated within endothelial cells to participate in this process. Further research is needed to determine whether PICALM expression is influenced by A&bgr; levels and whether it affects A&bgr; uptake and transport by endothelial cells.

Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity

Experimental reduction of neprilysin (NEP) or insulin-degrading enzyme (IDE) in vivo exacerbates &bgr;-amyloid accumulation in the brain. The level of these enzymes is reportedly reduced during aging and in postmortem brains of patients with sporadic Alzheimer disease (AD). To distinguish between primary decreases in NEP and IDE activity that might contribute to &bgr;-amyloid accumulation and decreases secondary to neurodegenerative changes in AD, we measured NEP and IDE levels by indirect sandwich ELISA and enzyme activities by immunocapture-based fluorogenic assays in postmortem frontal cortex from patients of different ages and at different pathological stages of AD, as indicated by Braak tangle stage. The ELISA measurements of neuron-specific enolase were used to adjust for neuronal loss. Both unadjusted and neuron-specific enolase-adjusted NEP levels and activity were significantly increased in AD and positively correlated with Braak stage but negatively with age in AD patients. Insulin-degrading enzyme activity was higher in AD than controls; this was significant after adjustment for neuron-specific enolase level; unadjusted IDE protein level was decreased in AD but not after adjustment. Our findings suggest that reduction in NEP and IDE activity is not the primary cause of &bgr;-amyloid accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

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.

Synaptic protein levels altered in vascular dementia

Cerebral ischaemia is the defining pathophysiological abnormality in most forms of vascular dementia (VAD), but the pathogenesis of the dementia remains poorly understood. In Alzheimers disease (AD), there is early loss of synaptic proteins, but these have been little studied in VAD.

Endothelin-converting enzyme-1 activity, endothelin-1 production, and free radical-dependent vasoconstriction in Alzheimer's disease

Alzheimers disease (AD) patients have reduced cerebral blood flow. This precedes dementia and may contribute to its progression. In mice that overexpress amyloid-β protein precursor, cerebral blood flow declines before the development of plaques or cognitive abnormalities. In the brain, endothelin-1 (ET-1) is a locally acting vasoconstrictor, produced in neurons by endothelin-converting enzyme (ECE)-2 and in endothelial cells by ECE-1. Both ECEs are also capable of cleaving amyloid-β (Aβ). We previously showed ECE-2 and ET-1 to be elevated in postmortem temporal cortex from AD patients, and ECE-2 expression and ET-1 release to be upregulated by Aβ42 in vitro. We have now studied isolated leptomeningeal blood vessels from postmortem brains and found that although ECE-1 level is reduced, ECE-1 activity and ET-1 level are significantly elevated in AD vessels. This is specific to AD as there is no specific change in vascular dementia vessels. In primary cultures of human brain endothelial cells, both Aβ40 and Aβ42 caused a significant increase in ET-1 release, the increase being particularly pronounced with Aβ40. In view of previous studies implicating free radicals in the endothelial dysfunction caused by Aβ40, we examined whether Aβ-mediated ET-1 release could be prevented by the antioxidant superoxide dismutase. Addition of superoxide dismutase to cells exposed to Aβ40 prevented the increase in the concentration of ET-1. Our findings indicate that cerebral vasoconstriction induced by Aβ results in part from a free radical-mediated increase in ECE-1 activity and ET-1 production.

Aβ degradation or cerebral perfusion? Divergent effects of multifunctional enzymes

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

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.

Cerebral Aβ40 and systemic hypertension

Mid-life hypertension and cerebral hypoperfusion may be preclinical abnormalities in people who later develop Alzheimer’s disease. Although accumulation of amyloid-beta (Aβ) is characteristic of Alzheimer’s disease and is associated with upregulation of the vasoconstrictor peptide endothelin-1 within the brain, it is unclear how this affects systemic arterial pressure. We have investigated whether infusion of Aβ40 into ventricular cerebrospinal fluid modulates blood pressure in the Dahl salt-sensitive rat. The Dahl salt-sensitive rat develops hypertension if given a high-salt diet. Intracerebroventricular infusion of Aβ induced a progressive rise in blood pressure in rats with pre-existing hypertension produced by a high-salt diet (p < 0.0001), but no change in blood pressure in normotensive rats. The elevation in arterial pressure in high-salt rats was associated with an increase in low frequency spectral density in systolic blood pressure, suggesting autonomic imbalance, and reduced cardiac baroreflex gain. Our results demonstrate the potential for intracerebral Aβ to exacerbate hypertension, through modulation of autonomic activity. Present findings raise the possibility that mid-life hypertension in people who subsequently develop Alzheimer’s disease may in some cases be a physiological response to reduced cerebral perfusion complicating the accumulation of Aβ within the brain.