Studies suggest that activation of phosphoinositide 3-kinaseAkt 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: GSK3b 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.

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The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of ϐ-amyloid (Aϐ) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive Aϐ to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.

The Prion protein is a cell surface glycoprotein expressed by neurons, glial cells and several peripheral cells. The conversion of P r p to PrPsc during prion replication appears to take place along an endocytic pathway. In this study, we investigate the localisation and relative levels of P~P' in various cell types, along with it's trafficking via R~M positive early endosomes using the HelaRaMGFP cell line.We have tested a number of cell types with a panel of antibodies including PRI 308. 3F4. SAF 15 and SAF 32 for localisation by immunofluorescence and for levels of expression by immunoblotting. The cell lines tested include, A431; an epidermal carcinoma cell line, SH-SY-5Y; a neuroblastoma cell line and Jurkatts; lymphocytes derived from human leukemia cells and a stable HeLa cell line expressing the Rab4 small GTPase tagged to the green fluorescent protein (Rab4GFP).Results obtained from immunofluorescence experiments for all cell lines showed that PRI 308 (2mg/ml) displays the greatest signal extensively on the plasma membrane. The 3F4 antibody (2.5mg/ml) gave a less intense signal while SAF 15 and SAF 32 (both at 20mg/ml) resulted in no signal. Confocal microscopy was used to investigate colocalisation of the Rab4 protein with the F'rion protein using HelaRab4GFP cell line. Of the four antibodies tested, the only antibody which resulted in a detectable signal by Western blotting was SAF 15 at a concentration of IOmg/ml. By immunoblotting with this antibody we find that all cell lines tested expressed PrP at detectable levels. Results will be presented comparing relative levels of PrP expression as well as the biochemical complexity in these cell types. Correspondance to M. McCaffrey : e-mail; m.mccaffrey@ucc.ie 45 Enrichment OF fall length prcsenilin (PS) proteins in hnman b b solnble h c t i o n s : redneed levels of PS-2 in these fractions in Alzheimer's disepar brain College, Lee Maltings, Cork, Ireland -Mutations in the genes encoding presedin proteins (PSI and PS-2 ) are linked to familial Alzbeimefs disease. Functionally PS-Ihas been strongly associated with kcatenin. We detected highly significant reductions in soluble pools of p-catenin in Alzheimer's disease (AD) brain compared to control brain. This led us to investigate expression levels of PS proteins in particulate and soluble fractions (100,ooO g, l h r spin) from the same AD (n=8) and control (n=8) brains. The integrity of the particulate /soluble separation was monitored using calnexin, and insulin like growth factor receptor which were detected exclusively in particulate fractions. PS-1 N antisem detected full-length protein of 48kDa and N-terminal fragments (NTF) of 28-36 KDa in control and AD homogenates with higher expression levels of NTF than full length protein observed. Examination of particulate and soluble fractions with PS-1 N terminal antibody surprisingly revealed high levels of full-length PS-1 protein and no N T F s in the soluble ftactions, with the particulate fractions containing only NTFs. Levels of the 2SkDa NTF were d u ...

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