Alzheimer’s disease is a neurodegenerative disorder which has been characterised with genetic (apolipoproteins), protein (ß-amyloid and tau) and lipid oxidation/metabolism alterations in its pathogenesis. In conjunction with the Dementia Research Group, Bristol University, investigation into genetic, protein and lipid oxidation in Alzheimer’s disease was conducted. A large sample cohort using the double-blind criteria, along with various clinical and chemical data sets were used to improve the statistical analysis and therefore the strength of this particular study. Bristol University completed genetic and protein analysis with lipid oxidation assays performed at Aston University. Lipid oxidation is a complex process that creates various biomarkers, from transient intermediates, to short carbon chain products and cyclic ring structures. Quantification of these products was performed on lipid extracts of donated clinical diseased and non-diseased frontal and temporal brain regions, from the Brain Bank within Frenchay Hospital. The initial unoxidised fatty acids, first transient oxidation intermediates the conjugated dienes and lipid hydroperoxides, the endpoint aldehyde biomarkers and finally the cyclic isoprostanes and neuroprostanes were determined to investigate lipid oxidation in Alzheimer’s. Antioxidant levels were also investigated to observe the effect of oxidation on the defence pathways. Assays utilised in this analysis included; fatty acid composition by GC-FID, conjugated diene levels by HPLC-UV and UV-spec, lipid hydroperoxide levels by FOX, aldehyde content by TBARs, antioxidant status by TEAC and finally isoprostane and neuroprostane quantification using a newly developed EI-MS method. This method involved the SIM of specific ions from F-ring isoprostane and neuroprostane fragmentation, which enabled EI-MS to be used for their quantification. Analyses demonstrated that there was no significant difference between control and Alzheimer samples across all the oxidation biomarkers for both brain regions. Antioxidants were the only marker that showed a clear variance; with Alzheimer samples having higher levels than the age matched controls. This unique finding is supported with the observed lower levels of lipid oxidation biomarkers in Alzheimer brain region samples. The increased antioxidant levels indicate protection against oxidation which may be a host response to counteract the oxidative pathways, but this requires further investigation. In terms of lipid oxidation, no definitive markers or target site for therapeutic intervention have been revealed. This study concludes that dietary supplementation of omega-3 fatty acids or antioxidants would most likely be ineffective against Alzheimer disease, although it may support improvement in other areas of general health.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. Amyloid precursor protein (APP) processing and the subsequent generation of amyloid β (Aβ) are central to the pathogenesis of AD, as soluble, oligomeric Aβ peptides are thought to be the toxic species driving disease progression. Flavonoids, a group of dietary polyphenols, have been shown to possess cognitive health benefits. Epidemiological evidence suggests they could play a role in risk reduction in dementia. In vitro and in vivo reports suggest flavonoids can modulate APP metabolism and Aβ production, although the most effective compounds and the underlying mechanism of action remain unclear. This study identified select flavonoids that were able to reduce amyloidogenic processing in primary cortical neurons at physiologically relevant concentrations. An APP-Gal4 gene reporter assay was characterised for identification of modulators of APP processing in primary neurons. It was tested under physiological conditions, in a pro-oxidant environment or against pathological levels of Aβ production through introduction of a known familial AD (FAD) causing mutation of APP (K595N/M596L). Using this system, five flavonoids were identified that inhibited amyloidogenic APP processing at 100 nM, these were: fisetin, pelargonidin, sinensetin, (-) epicatechin and epigallocatechin. Due to known bioavailability, the catechin family were investigated further and epigallocatechin and (-) epicatechin were confirmed as potent (low nanomolar) inhibitors of amyloidogenic processing. Investigation into the mechanism of action by (-) epicatechin suggested it was likely through indirect BACE1 activity inhibition, independent of BACE1 expression. In addition to activity at APP processing (-) epicatechin also induced extracellular signal-regulated kinase (ERK) activation and transcriptional activity. Investigation of other lead flavonoids also identified fisetin as an inhibitor of ERK-dependent transcriptional activity. This study supports the hypothesis that flavonoids are modulators of APP processing and that they could offer a potential therapy for the prophylaxis of AD.

The aggregation and deposition of ß-amyloid (Aß) in the brain has long been implicated in the neurotoxic pathways causing Alzheimer's disease (AD). Recent data suggests that early, soluble oligomers of Aß are the toxic species. Oxidative stress may be partly responsible for the toxicity of the peptide as hydrogen peroxide (H2O2)has been found to be produced during the early stages of aggregation.

Alzheimer's disease (AD) is thought to be initiated by the abnormal accumulation of Aß within the brain. Cerebrovascular dysfunction and cerebral hypoperfusion are central to the development vascular dementia (VaD) but are also thought to contribute to the progression of AD. A reduction in cerebral perfusion can be demonstrated prior to the onset of dementia in people who subsequently develop AD, and in patients with early disease the extent of reduction in perfusion predicts the rate of cognitive decline. In AD, there is also reported to be oxidative stress in the brain and it has been posited that this may serve as a pathogenic link between Aß, endothelial dysfunction and neuronal damage. Recent studies have also implicated the endotheJin pathway in the endothelial dysfunction in AD. In this pathway the production of endothelin-l (ET-1), a potent mediator of vasoconstriction, is catalysed by endothelin-converting enzyme (ECE)-1 in endothelial cells and ECE-2 in neurons. Both endothelin-converting enzymes are capable of degrading Aß and ECE-2 expression is upregulated by Aß.

The cholinergic neurons of the basal forebrain are vulnerable in Alzheimer's disease (AD). These neurons require trophic support by nerve growth factor (NGF) and, uniquely in the brain, express both NGF receptors: TrkA and p75NTR. Reduced support by NGF has been associated with neuronal loss and formation of amyloid plaques and neurofibrillary tangles. This study aimed to increase understanding of proNGF trophic signalling via TrkA/p75NTR and its impact on cholinergic survival in sporadic AD.

Alzheimer's disease (AD) is characterised by cognitive dysfunction and the appearance of ?-amyloid plaques (Ap), localised neuroinflammation and neuronal loss throughout the brain. These features are predominantly found in regions important to cognitive function such as the frontal cortex (FC) and hippocampus. The locus coeruleus (LC) is a main source of noradrenaline (NA) in the brain and released NA plays an important role modulating cognitive processes and may also regulate the inflammatory response. LC degeneration is apparent in the early stages of AD, as are decreased NA concentrations in LC projections regions. The relationship between NA dysfunction and AD pathogenesis remains unclear.

Amyloid beta protein (AP) is found in elevated levels in the brains of individuals with Alzheimer's disease, the increased levels of AP are thought to be a causative factor in the pronounced cortical atrophy that is observed post-mortem. Application of exogenous Apuo at physiological and pathological concentrations did not induce cell death in SH-SY5Y cells, differentiated SH-SY5Y cells nor in primary ortical neurone cultures when viability was measured using the Live/Dead cytotoxicity assay. Therefore, rather than a cytotoxic role of Ap this data suggests a more neuroprotective role.

Alzheimer's disease (AD) is the most common form of dementia. The main pathological hallmark associated with the disease is the presence of senile plaques that are composed of amyloid beta peptide (Aβ). This peptide is a key component in disease pathology and is thought to underlie neuronal loss seen in AD brains. Recent evidence now suggests a physiological role for the peptide in the maintenance of neuronal excitability. At physiological concentrations of Aβ (10 nM), we have previously demonstrated the modulation of Kv4.2 channels. In the present study we investigated both the physiological and the pathological concentrations of Aβ that contains 40 amino acid residues (Aβ 1-40), and determined its effects on Kv4.2 channels. Aβ1-40 was toxic to cerebellar granule neurons at a concentration of 1 μM and above, therefore this concentration was used to examine pathological effects. Physiological effects were examined using 10 nM Aβ1-40 . Significant reductions in A-type K+ channel current in hippocampal and cerebellar granule neurons was observed at 1μM, in a Ca2+ dependent manner. Cellular localisation of Kv4.2 was determined using immunofluorescence Aβ1-40 reduced both Kv4.2 and postsynaptic density 95 (PSD-95), at hippocampal dendrites at 1μ M. At 10 nM, Kv4.2 channel expression was increased independently of PSD-95. In Kv4.2 overexpressing cell lines, Aβ1-40 had no modulatory effect at the pathological concentration. The data presented herein demonstrate a reduction in K" channel current in neurons in response to soluble Aβ1-40 (1 μM). Furthermore, we show a modulatory effect of Aβ1 -40 on Kv4.2 expression in hippocampal dendrites at both pathological and physiological concentrations. PSD-95 expression was only co-modulated at the pathological concentrations, indicating divergent mechanisms dependent on the v concentrations of Aβ1-40 used. The loss of Kv4.2 and PSO-95 at 1μM Aβ1-40 could possibly correlate with early synaptic dysfunction in Alzheimer's Disease

Alzheimer's disease (AD), a progressive, neurodegenerative brain disorder, is an impending socio-economic crisis due to the aging global population. Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction and memory impairments which underlie AD, but their mechanisms of action are poorly understood. Recently, the cellular prion protein (Prpc) was identified as a high affinity receptor which mediates the neuronal binding and memory impairments of AI3 oligomers. In this study, the cellular biology of the interaction between AI3 oligomers and Prpc was investigated. We report that fibrillar Aβ oligomers recognised by the QC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to neuronal cells expressing Prpc. The green tea polyphenol (-)-epigallocatechin gallate (EGCG) and the red wine extract resveratrol both re-modelled the fibrillar conformation of AI3 oligomers. The resulting non-fibrillar oligomers displayed significantly reduced binding to Prpc-expressing cells and were no longer cytotoxic. Fluorescence microscopy and co-localisation with sub-cellular markers revealed that the AI3 oligomers co-internalised with Prpc, accumulated in endosomes and subsequently trafficked to Iysosomes. We report that the cell surface binding, internalisation and downstream toxicity of Aβ oligomers is dependent on the transmembrane low density lipoprotein receptor- related protein-1 (LRP1). Western blotting revealed that the binding of AI3 oligomers to cell surface Prpc impaired its ability to inhibit the activity of the l3-secretase BACE1 which cleaves the amyloid precursor protein to produce AI3. Using an siRNA approach, AI3 oligomers were found to activate the signalling molecule STEP61 in a Prpc-dependent manner. AI3 oligomers also stimulated caspase-3 activation via Prpc. These data indicate that soluble, fibrillar Aβ oligomers bind to Prpc in a conformation-dependent manner and require the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets for therapeutic intervention in AD.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease that affects millions of people worldwide. AD is characterised by the accumulation aggregation and deposition within the brain of amyloid ~ (A~), a 40-42 residue peptide, and hyperphosphorylated Tau. A~ is a cleavage product of the amyloid precursor protein (APP) produced by the sequential cleavage of the ~-secretase, 8ACE1 (~-sjte APP Cleaving enzyme), and the v-secretase. APP can also be proteolytically processed by an alternative pathway where an a-secretase cleaves APP within the A~ region followed by v-cleavage. This precludes the formation of A~. Protein-protein interactions play an important role in regulating protein function; elucidating how the interactions between APP and other proteins affect A~ accumulation in an age-dependent manner is crucial to understanding AD progression. The Apolipoprotein E receptor 2 (ApoEr2) interacts with APP and alters its cleavage, though its exact effect is unclear as it has been shown to both increase and decrease ~-secretase cleavage of APP. To understand the reason for this disparity, 3 different splice variants of ApoEr2 have been expressed in HEK cells and their effects on the levels of APP cleavage products assessed. The isoforms chosen were a full length form (ApoEr2 FL), a form lacking the exon 5 and 15 encoded regions (ApoEr2 1:::.5,15), and a form lacking exon 18 (ApoEr2 1118) which encodes a cytoplasmic proline rich domain. These were chosen as all 3 isoforrns are brain expressed and have been used by different groups in investigating the effects of ApoEr2 on APP processing. It was found that 2 isoforms of ApoEr2 (ApoEr2 FL and 1:::.5,15) caused a reduction in ~- and a-cleavage of APP of -50%, where as ApoEr2 l::.18 increased ~-cleavage -200% whilst having no effect on a- cleavage, To identify the mechanism behind the variation between the isoforms effect on APP Cleavage, the post translational processing of ApoEr2 has been investigated, ApoEr2 was identified as being both N- and O-glycosylated and differences between the isoforms of ApoEr2 in the extent of fully glycosylatyed protein were identified. ApoEr2 l::.5,15 was produced as a fully glycosylated protein. With ApoEr2 FL and 1118 a partially glycosylated protein is also detectable at -25% of total ApoEr2 1:.18 and 40% of total ApoEr2 FL ApoEr2 is also shed from the plasma membrane and there were differences in the shedding of the isoforms of ApoEr2, with ApoEr2 1:.5,15 shed to a lower level than ApoEr2 FL or 1:::.18. The enzyme responsible for the shedding of ApoEr2 has been identified as ADAM10, an enzyme also involved in cleaving APP. The subcellular localisation of ApoEr2 was also investigated and increased detection of ApoEr2 in the early endosome may explain the isoform specific effect of ApoEr2 on APP cleavage. This work has shown for the first time that different splice forms of ApoEr2 have radically different effects on APP proteolytic processing, The fact that splice forms of a protein can have such different effects on APP processing shows that the regulation of APP cleavage is exceedingly complex. This work has also significantly extended what is known about the post translational processing of ApoEr2 with the identification of the enzyme responsible for the shedding of ApoEr2 and has shown that there is substantial variation in post translational processing of the different isoforms of ApoEr2.