Platelet Membrane ß-Secretase Activity in Mild Cognitive Impairment and Conversion to Dementia: a Longitudinal Study.

A blood-based biomarker to complement the clinical and neuropsychological assessments used to evaluate the risk of individuals with mild cognitive impairment (MCI) developing Alzheimer's disease (AD) would be invaluable. Previous pilot studies by our group identified elevated platelet membrane ß-secretase activity in patients with AD and MCI, as compared to controls, and this activity was influenced by membrane cholesterol levels. The present study investigated baseline platelet membrane ß-secretase activity and cholesterol levels in 97 MCI participants and 85 controls and explored whether these parameters differed in individuals with stable MCI, as compared to those who subsequently developed AD. To evaluate signal specificity, ß-secretase activity assays were conducted in the presence and absence of beta-site amyloid-ß protein precursor-cleaving enzyme (BACE) inhibitors. Baseline platelet membrane ß-secretase activity did not differ significantly in MCI participants, as compared to controls, and platelet membrane cholesterol levels were significantly lower in the MCI group. The longitudinal study indicated that the activities inhibited by two different BACE inhibitors did not predict conversion to AD; however, the activity that was not affected by BACE inhibitors was significantly (40%) higher in individuals with stable MCI, as compared with those who subsequently developed AD. These findings indicated that further research into the source of this activity could contribute to a measure facilitating prediction of the risk of conversion from MCI to AD.

Alleles that increase risk for type 2 diabetes mellitus are not associated with increased risk for Alzheimer's disease.

Although epidemiological studies suggest that type 2 diabetes mellitus (T2DM) increases the risk of late-onset Alzheimer's disease (LOAD), the biological basis of this relationship is not well understood. The aim of this study was to examine the genetic comorbidity between the 2 disorders and to investigate whether genetic liability to T2DM, estimated by a genotype risk scores based on T2DM associated loci, is associated with increased risk of LOAD. This study was performed in 2 stages. In stage 1, we combined genotypes for the top 15 T2DM-associated polymorphisms drawn from approximately 3000 individuals (1349 cases and 1351 control subjects) with extracted and/or imputed data from 6 genome-wide studies (>10,000 individuals; 4507 cases, 2183 controls, 4989 population controls) to form a genotype risk score and examined if this was associated with increased LOAD risk in a combined meta-analysis. In stage 2, we investigated the association of LOAD with an expanded T2DM score made of 45 well-established variants drawn from the 6 genome-wide studies. Results were combined in a meta-analysis. Both stage 1 and stage 2 T2DM risk scores were not associated with LOAD risk (odds ratio = 0.988; 95% confidence interval, 0.972-1.004; p = 0.144 and odds ratio = 0.993; 95% confidence interval, 0.983-1.003; p = 0.149 per allele, respectively). Contrary to expectation, genotype risk scores based on established T2DM candidates were not associated with increased risk of LOAD. The observed epidemiological associations between T2DM and LOAD could therefore be a consequence of secondary disease processes, pleiotropic mechanisms, and/or common environmental risk factors. Future work should focus on well-characterized longitudinal cohorts with extensive phenotypic and genetic data relevant to both LOAD and T2DM.

Plasma clusterin levels and the rs11136000 genotype in individuals with mild cognitive impairment and Alzheimer's disease.

AIM: Substantial evidence links atherosclerosis and Alzheimer's disease (AD). Apolipoproteins, such as apolipoprotein E, have a causal relationship with both diseases. The rs11136000 SNP within the CLU gene, which encodes clusterin (apolipoprotein J), is also associated with increased AD risk. The aim of this study was to investigate the relationship between plasma clusterin and the rs11136000 genotype in mild cognitive impairment (MCI) and AD. METHODS: Plasma and DNA samples were collected from control, MCI and AD subjects (n=142, 111, 154, respectively). Plasma clusterin was determined by ELISA and DNA samples were genotyped for rs11136000 by TaqMan assay. RESULTS: Plasma clusterin levels were higher in MCI and AD subjects vs. controls (222.3 ± 61.3 and 193.6 ± 58.2 vs. 178.6 ± 52.3 µg/ml, respectively; p<0.001 for both comparisons), and in MCI vs. AD (p<0.05). Plasma clusterin was not influenced by genotype in the MCI and AD subjects, although in control subjects plasma clusterin was lower in the TT vs. TC genotypes (157.6 ± 53.4 vs. 188.6 ± 30.5 µg/ml; p<0.05). CONCLUSION: This study examined control, MCI and AD subjects, identifying for the first time that plasma clusterin levels were influenced, not only by the presence of AD, but also the transitional stage of MCI, while rs11136000 genotype only influenced plasma clusterin levels in the control group. The increase in plasma clusterin in MCI and AD subjects may occur in response to the disease process and would be predicted to increase binding capacity for amyloid-beta peptides in plasma, enhancing their removal from the brain.

The development of effective biomarkers for Alzheimer's disease: a review.

OBJECTIVE: There is a widely recognised need to develop effective Alzheimer's disease (AD) biomarkers to aid the development of disease-modifying treatments, to facilitate early diagnosis and to improve clinical care. This overview aims to summarise the utility of key neuroimaging and cerebrospinal fluid (CSF) biomarkers for AD, before focusing on the latest efforts to identify informative blood biomarkers. DESIGN: A literature search was performed using PubMed up to September 2011 for reviews and primary research studies of neuroimaging (magnetic resonance imaging, magnetic resonance spectroscopy, positron emission tomography and amyloid imaging), CSF and blood-based (plasma, serum and platelet) biomarkers in AD and mild cognitive impairment. Citations within individual articles were examined to identify additional studies relevant to this review. RESULTS: Evidence of AD biomarker potential was available for imaging techniques reflecting amyloid burden and neurodegeneration. Several CSF measures are promising, including 42 amino acid ß-amyloid peptide (Aß42 ); total tau (T-tau) protein, reflecting axonal damage; and phosphorylated tau (P-tau), reflecting neurofibrillary tangle pathology. Studies of plasma Aß have produced inferior diagnostic discrimination. Alternative plasma and platelet measures are described, which represent potential avenues for future research. CONCLUSIONS: Several imaging and CSF markers demonstrate utility in predicting AD progression and determining aetiology. These require standardisation before forming core elements of diagnostic criteria. The enormous potential available for identifying a minimally-invasive, easily-accessible blood measure as an effective AD biomarker currently remains unfulfilled.

α-Synuclein mRNA and soluble α-synuclein protein levels in post-mortem brain from patients with Parkinson's disease, dementia with Lewy bodies, and Alzheimer's disease.

α-Synuclein is a neuronal protein implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Whilst increased α-synuclein expression due to gene duplication or triplication can cause familial PD, previous studies of α-synuclein levels in idiopathic disease have produced conflicting data. We quantified α-synuclein mRNA and soluble protein in five human post-mortem brain regions from four groups of individuals with PD, DLB, Alzheimer's disease (AD) and matched controls. α-Synuclein mRNA levels, measured using quantitative real-time PCR, did not differ significantly between groups in any brain regions examined. In contrast, levels of soluble α-synuclein protein, measured by ELISA, were significantly lower in 4 of the 5 regions for patients with DLB, and in 2 of the 5 regions for patients with PD, compared to controls. Soluble α-synuclein protein levels were not significantly different in the AD patients, compared to controls, in 4 of the 5 regions. This study indicates that although levels of soluble α-synuclein protein are lower in DLB and PD, there is no evidence for a corresponding decrease in α-synuclein mRNA levels. This might result from altered translation, or removal of α-synuclein protein from a soluble detectable state, either by turnover or conversion to an insoluble form.

Genetic variation in MME in relation to neprilysin protein and enzyme activity, Aß levels, and Alzheimer's disease risk.

Neprilysin (NEP), also known as membrane metalloendopeptidase (MME), is considered amongst the most important ß-amyloid (Aß)-degrading enzymes with regard to prevention of Alzheimer's disease (AD) pathology. Variation in the NEP gene (MME) has been suggested as a risk factor for AD. We conducted a genetic association study of 7MME SNPs - rs1836914, rs989692, rs9827586, rs6797911, rs61760379, rs3736187, rs701109 - with respect to AD risk in a cohort of 1057 probable and confirmed AD cases and 424 age-matched non-demented controls from the United Kingdom, Italy and Sweden. We also examined the association of these MME SNPs with NEP protein level and enzyme activity, and on biochemical measures of Aß accumulation in frontal cortex - levels of total soluble Aß, oligomeric Aß(1-42), and guanidine-extractable (insoluble) Aß - in a sub-group of AD and control cases with post-mortem brain tissue. On multivariate logistic regression analysis one of the MME variants (rs6797911) was associated with AD risk (P = 0.00052, Odds Ratio (O.R. = 1.40, 95% confidence interval (1.16-1.70)). None of the SNPs had any association with Aß levels; however, rs9827586 was significantly associated with NEP protein level (p=0.014) and enzyme activity (p=0.006). Association was also found between rs701109 and NEP protein level (p=0.026) and a marginally non-significant association was found for rs989692 (p=0.055). These data suggest that MME variation may be associated with AD risk but we have not found evidence that this is mediated through modification of NEP protein level or activity.

Zinc transporter mRNA levels in Alzheimer's disease postmortem brain.

Zinc (Zn2+) is concentrated into pre-synaptic vesicles and co-released with neurotransmitter at some synapses. Zn2+ can accelerate assembly of the amyloid-ß peptides (Aß) and tau protein central to the neuropathological changes found in Alzheimer's disease (AD). Altered protein levels of the membrane Zn2+ transporters ZnT1, ZnT4, and ZnT6 have been reported in AD postmortem brain tissue. The present study analyzed mRNA levels of five established (LIV1, ZIP1, ZnT1, ZnT4, and ZnT6) and one potential (PRNP) Zn2+ transporter in human postmortem brain tissue from Braak-staged individuals with AD and controls using quantitative real-time PCR. Four cortical regions (middle temporal gyrus, superior occipital gyrus, superior parietal gyrus, and superior frontal gyrus) and cerebellum were examined. PRNP mRNA levels were decreased by ∼30% in all four cortical regions examined in AD patients, but unchanged in the cerebellum. In contrast, some increases in mRNA levels of the other more established Zn2+ transporters (LIV1, ZIP1, ZnT1, ZnT6) were found in AD cortex. The ratios of the mRNA levels of LIV1, ZIP1, ZnT1, ZnT4, and ZnT6/mRNA level of neuron specific enolase increased significantly as the disease progressed and Braak stage increased. Significant correlations were also identified between mRNA levels of several of the Zn2+ transporters investigated. These expression changes could either reflect or cause the altered cortical Zn2+ distribution in AD, potentially increasing the likelihood of interactions between Zn2+ and Aß or tau protein.

The role of variation at AßPP, PSEN1, PSEN2, and MAPT in late onset Alzheimer's disease.

Rare mutations in AßPP, PSEN1, and PSEN2 cause uncommon early onset forms of Alzheimer's disease (AD), and common variants in MAPT are associated with risk of other neurodegenerative disorders. We sought to establish whether common genetic variation in these genes confer risk to the common form of AD which occurs later in life (>65 years). We therefore tested single-nucleotide polymorphisms at these loci for association with late-onset AD (LOAD) in a large case-control sample consisting of 3,940 cases and 13,373 controls. Single-marker analysis did not identify any variants that reached genome-wide significance, a result which is supported by other recent genome-wide association studies. However, we did observe a significant association at the MAPT locus using a gene-wide approach (p = 0.009). We also observed suggestive association between AD and the marker rs9468, which defines the H1 haplotype, an extended haplotype that spans the MAPT gene and has previously been implicated in other neurodegenerative disorders including Parkinson's disease, progressive supranuclear palsy, and corticobasal degeneration. In summary common variants at AßPP, PSEN1, and PSEN2 and MAPT are unlikely to make strong contributions to susceptibility for LOAD. However, the gene-wide effect observed at MAPT indicates a possible contribution to disease risk which requires further study.

No evidence that extended tracts of homozygosity are associated with Alzheimer's disease.

We sought to investigate the contribution of extended runs of homozygosity in a genome-wide association dataset of 1,955 Alzheimer's disease cases and 955 elderly screened controls genotyped for 529,205 autosomal single nucleotide polymorphisms. Tracts of homozygosity may mark regions inherited from a common ancestor and could reflect disease loci if observed more frequently in cases than controls. We found no excess of homozygous tracts in Alzheimer's disease cases compared to controls and no individual run of homozygosity showed association to Alzheimer's disease.

BACE1 mRNA expression in Alzheimer's disease postmortem brain tissue.

ß-site AßPP cleaving enzyme 1 (BACE1) catalyses the rate-limiting step for production of amyloid-ß (Aß) peptides, involved in the pathological cascade underlying Alzheimer's disease (AD). Elevated BACE1 protein levels and activity have been reported in AD postmortem brains. Our study explored whether this was due to elevated BACE1 mRNA expression. RNA was prepared from five brain regions in three study groups: controls, individuals with AD, and another neurodegenerative disease group affected by either Parkinson's disease (PD) or dementia with Lewy bodies (DLB). BACE1 mRNA levels were measured using quantitative realtime PCR (qPCR) and analyzed by qbasePLUS using validated stably-expressed reference genes. Expression of glial and neuronal markers (glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), respectively) were also analyzed to quantify the changing activities of these cell populations in the tissue. BACE1 mRNA levels were significantly elevated in medial temporal and superior parietal gyri, compared to the PD/DLB and/or control groups. Superior frontal gryus BACE1 mRNA levels were significantly increased in the PD/DLB group, compared to AD and control groups. For the AD group, BACE1 mRNA changes were analyzed in the context of the reduced NSE mRNA, and strongly increased GFAP mRNA levels apparent as AD progressed (indicated by Braak stage). This analysis suggested that increased BACE1 mRNA expression in remaining neuronal cells may contribute to the increased BACE1 protein levels and activity found in brain regions affected by AD.

ZnT3 mRNA levels are reduced in Alzheimer's disease post-mortem brain.

BACKGROUND: ZnT3 is a membrane Zn(2+ )transporter that is responsible for concentrating Zn(2+ )into neuronal presynaptic vesicles. Zn(2+ )homeostasis in the brain is relevant to Alzheimer's disease (AD) because Zn(2+ )released during neurotransmission may bind to Abeta peptides, accelerating the assembly of Abeta into oligomers which have been shown to impair synaptic function. RESULTS: We quantified ZnT3 mRNA levels in Braak-staged human post mortem (pm) brain tissue from medial temporal gyrus, superior occipital gyrus, superior parietal gyrus, superior frontal gyrus and cerebellum from individuals with AD (n = 28), and matched controls (n = 5) using quantitative real-time PCR. ZnT3 mRNA levels were significantly decreased in all four cortical regions examined in the AD patients, to 45-60% of control levels. This reduction was already apparent at Braak stage 4 in most cortical regions examined. Quantification of neuronal and glial-specific markers in the same samples (neuron-specific enolase, NSE; and glial fibrillary acidic protein, GFAP) indicated that loss of cortical ZnT3 expression was more pronounced, and occurred prior to, significant loss of NSE expression in the tissue. Significant increases in cortical GFAP expression were apparent as the disease progressed. No gene expression changes were observed in the cerebellum, which is relatively spared of AD neuropathology. CONCLUSIONS: This first study to quantify ZnT3 mRNA levels in human pm brain tissue from individuals with AD and controls has revealed a significant loss of ZnT3 expression in cortical regions, suggesting that neuronal cells in particular show reduced expression of ZnT3 mRNA in the disease. This suggests that altered neuronal Zn(2+ )handling may be an early event in AD pathogenesis.

Variation in RTN3 and PPIL2 genes does not influence platelet membrane beta-secretase activity or susceptibility to alzheimer's disease in the northern Irish population.

Beta-site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of beta-amyloid peptides (Abeta), which are proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (beta-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulates BACE1 expression. The present study investigated whether there was any association between genetic variation in RTN3 and PPIL2, and either risk for AD, or levels of platelet beta-secretase activity, in a large Northern Irish case-control sample. Four hundred and sixty-nine patients with a diagnosis of probable AD (NINCDS-ADRDA criteria) and 347 control individuals (MMSE > 28/30) were genotyped. SNPs in both genes were selected by downloading genotype data from the International HapMap Project (Phase II) and tags selected using multimarker approach in Haploview, where r (2) > 0.8 and LOD > 3.0. Non-synonymous SNPs of interest were also included. Genotyping was performed by Sequenom iPLEX and TaqMan technologies. Alleles, genotypes and multi-marker haplotypes were tested for association with AD, and platelet beta-secretase activities were measured for a subset of individuals (n = 231). Eight SNPs in RTN3 and 7 in PPIL2 were genotyped. We found no significant associations between allele, genotype or haplotype frequencies and risk of AD. Further, there was no effect of genotype on platelet membrane beta-secretase activity. We conclude that common or potentially functional genetic variation in these BACE1 interacting proteins does not affect platelet membrane beta-secretase activity or contribute to risk of AD in this population.

Reduced estimated glomerular filtration rate in Alzheimer's disease.

OBJECTIVES: Renal disease is increasingly regarded as an independent risk factor for vascular disease which in itself is believed to influence risk of AD. Alterations in amyloid homeostasis via reduced renal clearance of peripheral beta-amyloid (A|*beta*|) may represent another potential role for variation in renal function leading to increased risk of AD. We sought to examine estimates of glomerular filtration rate in AD and control groups. METHODS: AD patients were randomly recruited from the Memory Clinic of the Belfast City Hospital (n = 83). Genomic DNA was extracted from peripheral leucocytes and was genotyped for Apolipoprotein E using standard methods. Using creatinine values, age and gender, estimated Glomerular Filtration Rates (eGFR) were calculated using the isotope dilution mass spectrometry (IDMS)-traceable Modification of Diet in Renal Disease (MDRD) Study equation (using the United Kingdom National External Quality Assessment Scheme (UKNEQAS) correction factor). IDMS eGFR values were then compared between AD and control groups. RESULTS: Significant baseline differences in age, diastolic blood pressure, education level attained and APOE |*epsilon*|4 carriage were noted between cases and controls. The AD group had a significantly lower eGFR versus controls (69 vs 77 ml/min) which persisted after adjustment for possible confounders (p = 0.045). CONCLUSIONS: This case-control analysis suggests that using a relatively accurate estimate of renal function, patients with AD have greater renal impairment than cognitively normal controls. This may reflect impaired renal clearance of peripheral A|*beta*| or be a marker of shared vascular processes altering cerebral and renal functioning.

A novel reciprocal and biphasic relationship between membrane cholesterol and beta-secretase activity in SH-SY5Y cells and in human platelets.

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the Abeta peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane beta-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane beta-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH-SY5Y beta-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane beta-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.

Genetic variation in the alpha 7 nicotinic acetylcholine receptor is associated with delusional symptoms in Alzheimer's disease.

Psychotic symptoms are common in Alzheimer's disease (AD) and have a negative impact on quality of life. It is suggested that psychotic symptoms may be attributed to genetic risk factors which are revealed during neurodegeneration. CHRNA7, the gene for the alpha 7 nicotinic acetylcholine receptor, has been associated with schizophrenia in linkage and association studies. Hence we investigated single SNPs and haplotypes in CHRNA7 in relation to AD with psychosis in a large, well-characterised and previously described cohort within the Northern Ireland population. A significant association between delusions and the T allele of rs6494223 (P = 0.014, OR = 1.63, CI = 1.22-2.17) was found. This suggests that the alpha 7 receptor may be a suitable target for the treatment of AD with psychosis.

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue.

BACKGROUND: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. RESULTS: The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan(R) Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. CONCLUSION: This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

Familial genes in sporadic disease: common variants of alpha-synuclein gene associate with Parkinson's disease.

Genetic variation of the alpha-synuclein gene (SNCA) is known to cause familial parkinsonism, however the role of SNCA variants in sporadic Parkinson's disease (PD) remains elusive. The present study identifies an association of common SNCA polymorphisms with disease susceptibility in a series of Irish PD patients. There is evidence for association with alternate regions, of protection and risk which may act independently/synergistically, within the promoter region (Rep1; OR: 0.59, 95% CI: 0.37-0.84) and the 3'UTR of the gene (rs356165; OR: 1.67, 95% CI: 1.08-2.58). Given previous reports of association a collaborative effort is required which may exploit global linkage disequilibrium patterns for SNCA and standardise polymorphic markers used in each population. It is now crucial to identify the susceptibility allele and elucidate its functionality which may generate a therapeutic target for PD.

Clinical traits of LRRK2-associated Parkinson's disease in Ireland: a link between familial and idiopathic PD.

The role of genetics in parkinsonism has been confirmed over the last decade with the identification of genetic variation in seven genes, which are causative in familial forms of the disorder. A number of pathogenic mutations have been identified in the latest gene LRRK2, with a Gly2019Ser amino acid substitution identified in two siblings and one patient with idiopathic Parkinson's disease from Ireland. The clinical features resemble the idiopathic variant with a tremor predominant clinical picture shared by the siblings, slow progression of symptoms, and no observation of cognitive disturbance in all. The family and the sporadic individual were apparently not related and originated from different regions of Ireland, although haplotype analysis does suggest they share a common founder. The influence of the G2019S substitution on protein function and disease phenotype has yet to be fully resolved, but its elucidation will undoubtedly further our understanding of the mechanisms underlying Parkinson's disease.

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology.

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.

Proteasome-mediated effects on amyloid precursor protein processing at the gamma-secretase site.

Abeta (beta-amyloid) peptides are found aggregated in the cortical amyloid plaques associated with Alzheimer's disease neuropathology. Inhibition of the proteasome alters the amount of Abeta produced from APP (amyloid precursor protein) by various cell lines in vitro. Proteasome activity is altered during aging, a major risk factor for Alzheimer's disease. In the present study, a human neuroblastoma cell line expressing the C-terminal 100 residues of APP (SH-SY5Y-SPA4CT) was used to determine the effect of proteasome inhibition, by lactacystin and Bz-LLL-COCHO (benzoyl-Leu-Leu-Leu-glyoxal), on APP processing at the gamma-secretase site. Proteasome inhibition caused a significant increase in Abeta peptide levels in medium conditioned by SH-SY5Y-SPA4CT cells, and was also associated with increased cell death. APP is a substrate of the apoptosis-associated caspase 3 protease, and we therefore investigated whether the increased Abeta levels could reflect caspase activation. We report that caspase activation was not required for proteasome-inhibitor-mediated effects on APP (SPA4CT) processing. Cleavage of Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin), a caspase substrate, was reduced following exposure of SH-SY5Y-SPA4CT cells to lactacystin, and co-treatment of cells with lactacystin and a caspase inhibitor [Z-DEVD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)] resulted in higher Abeta levels in medium, augmenting those seen with lactacystin alone. This study indicated that proteasome inhibition could increase APP processing specifically at the gamma-secretase site, and increase release of Abeta, in the absence of caspase activation. This indicates that the decline in proteasome function associated with aging would contribute to increased Abeta levels.