The Amyloid Precursor Protein and Cell Viability in Diseases of the Ageing Brain.

The amyloid precursor protein (APP) has been extensively studied in relation to Alzheimer's disease, but the physiological function of the protein has yet to be determined. One possible role for APP may be to protect cells against the detrimental effects of excess copper. In the current study I have investigated which regions of the APP molecule participate in this putative function. I have used both Du145 and SH-SY5Y mammalian cell systems to explore this question, relating the generated results to both cancer and Alzheimer's Disease. Initial studies using western blotting showed that a range of prostate cancer cell lines express APP, and that increased APP expression levels may represent a more advanced stage of cancer progression. Exogenous copper altered the expression levels of APP in Du145 cells, immediately indicating the intricate relationship between APP and copper. Subsequently, using MTS assays, I found that APP can indeed enhance cell viability in the face of copper, in both of the cell systems stated above. This role is isoform dependent, being specific to the APP695 isoform, which is predominantly found in the brain. N-terminal copper binding and copper binding within the Abeta region of the APP holoprotein appeared to be a prerequisite for this action. The intracellular domain was also required in order to enhance cell viability, and the importance of this region was assigned to specific tyrosine residues within the domain. APP over-expression in Du145 cells also appeared to induce a partial epithelial to mesenchymal transition of the cells. Along with suggesting a possible physiological function for APP, these data suggest that prostate cancer epithelial cells metastasising to the brain might utilise APP to protect them against the increased copper levels found in this organ

The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers

Conversion to eslicarbazepine acetate monotherapy: A pooled analysis of 2 phase III studies.

OBJECTIVE: To assess the efficacy and safety of eslicarbazepine acetate (ESL) monotherapy. METHODS: This post hoc pooled analysis of 2 randomized double-blind studies (093-045 and -046) included adults with partial-onset seizures medically uncontrolled by 1 or 2 antiepileptic drugs (AEDs). Following the baseline period (8 weeks), eligible patients were randomized 2:1 to receive ESL 1,600 mg or 1,200 mg once daily for 18 weeks; the primary endpoint was study exit by meeting predefined exit criteria (signifying worsening seizure control). In each study, treatment was considered effective if the upper 95% confidence limit for exit rate was lower than the historical control threshold (65.3%). RESULTS: Pooled exit rates were as follows: ESL 1,600 mg = 20.6% (95% confidence interval: 15.6%-26.8%); ESL 1,200 mg = 30.8% (23.0%-40.5%). Use of 2 baseline AEDs or rescue medication, US location, epilepsy duration ≥20 years, and higher maximum baseline seizure frequency were associated with higher exit risks. Median percent reductions in standardized seizure frequency between baseline and the 18-week double-blind period were as follows: ESL 1,600 mg = 43.2%; ESL 1,200 mg = 35.7%; baseline carbamazepine use was associated with smaller reductions. Safety profiles were similar between ESL doses. CONCLUSIONS: Exit rates for ESL monotherapy (1,600 mg and 1,200 mg once daily) were lower than the historical control threshold, irrespective of baseline AED use and region, with no additional safety concerns identified. Clinical factors and location clearly influence treatment responses in conversion-to-monotherapy trials. CLASSIFICATION OF EVIDENCE: This pooled analysis provides Class IV evidence that for adults with medically uncontrolled partial-onset seizures, ESL monotherapy is well tolerated and effective

Zinc metalloproteinases and amyloid Beta-Peptide metabolism:the positive side of proteolysis in Alzheimer's disease

Alzheimer's disease is a neurodegenerative condition characterized by an accumulation of toxic amyloid beta- (Aβ-)peptides in the brain causing progressive neuronal death. Aβ-peptides are produced by aspartyl proteinase-mediated cleavage of the larger amyloid precursor protein (APP). In contrast to this detrimental "amyloidogenic" form of proteolysis, a range of zinc metalloproteinases can process APP via an alternative "nonamyloidogenic" pathway in which the protein is cleaved within its Aβ region thereby precluding the formation of intact Aβ-peptides. In addition, other members of the zinc metalloproteinase family can degrade preformed Aβ-peptides. As such, the zinc metalloproteinases, collectively, are key to downregulating Aβ generation and enhancing its degradation. It is the role of zinc metalloproteinases in this "positive side of proteolysis in Alzheimer's disease" that is discussed in the current paper

Charge exchange losses during cyclotron acceleration: experiment and theory

Quantitative estimates of charge exchange (CE) losses during acceleration are very important in the design and operation of heavy ion cyclotrons. Such estimates have been made using a vacuum model computer code which was developed to establish vacuum requirements for the MSU superconducting heavy ion cyclotron. This code uses pressure and cross-section data to calculate the radial loss of beam due to charge exchange. Since CE cross sections and radial pressure profiles are not always well known, certain specific measurements have been made using the LBL 88-Inch Cyclotron to provide experimental data needed to test the code. These include measurements of pressure versus radius under vacuum conditions closely approximating those existing during acceleration of /sup 14/N/sup 4 -/ and /sup 40/Ar/sup 8 -/ beams. Beam intensity versus radius data demonstrating transmission losses for three beams are presented. Comparisons with theoretical predictions are given

The E1 copper binding domain of full-length amyloid precursor protein promotes epithelial to mesenchymal transition in DU145 cells in isoform-specific manner

Epithelial to mesenchymal transition (EMT) confers migratory and dynamic properties on cells and, as such, plays a pivotal role in the development of metastatic, castration resistant prostate cancer. The amyloid precursor protein (APP), although most closely associated with the neurodegenerative condition Alzheimer's disease, has also been linked to the pathogenesis and prognosis of several cancers including prostate cancer. Aims: To investigate whether over-expression of APP could promote EMT in prostate cancer (PCa) DU145 cells and to determine the molecular prerequisites for this effect. Methodology: A range of APP molecular constructs were stably expressed in DU145 cells and their effects on EMT were monitored by morphological analysis and by immunoblotting for the EMT marker proteins, E-cadherin and vimentin. Results: Our results show that the full-length 695 amino acid isoform (APP695), but not APP751 or APP770, promoted EMT via a mechanism requiring an intact extracellular E1 copper binding domain and tyrosine687 within the cytosolic domain of the protein. Conclusion: Targeting the expression of APP695 or the E1 copper binding domain of the protein may, therefore, contribute to therapeutic strategies for the delay or prevention of prostate cancer metastasis

The histidine composition of the amyloid-β domain, but not the E1 copper binding domain, modulates β-secretase processing of amyloid-β protein precursor in Alzheimer's disease

Amyloid-β protein precursor (AβPP) proteolysis by β- and γ-secretases generates neurotoxic amyloid-β (Aβ)-peptides in Alzheimer's disease (AD). We have investigated the role of histidine residues within the extracellular E1 copper binding and Aβ domains of AβPP in its proteolysis. By stably expressing histidine to alanine AβPP mutant constructs in SH-SY5Y cells, we show that mutations in the E1 copper binding domain had no impact on α- or β-secretase processing. Mutation of histidine 14 within the Aβ-domain specifically down-regulated β-secretase processing without impacting on non-amyloidogenic proteolysis. Understanding how histidine 14 participates in AβPP proteolysis may reveal new intervention points for AD treatments

The amyloid precursor protein represses expression of acetylcholinesterase in neuronal cell lines

The toxic role of amyloid β peptides in Alzheimer's disease is well documented. Their generation is via sequential β- and γ-secretase cleavage of the membrane-bound amyloid precursor protein (APP). Other APP metabolites include the soluble ectodomains sAPPα and sAPPβ and also the amyloid precursor protein intracellular domain (AICD). In this study, we examined whether APP is involved in the regulation of acetylcholinesterase (AChE), which is a key protein of the cholinergic system and has been shown to accelerate amyloid fibril formation and increase their toxicity. Overexpression of the neuronal specific isoform, APP695, in the neuronal cell lines SN56 and SH-SY5Y substantially decreased levels of AChE mRNA, protein, and catalytic activity. Although similar decreases in mRNA levels were observed of the proline-rich anchor of AChE, PRiMA, no changes were seen in mRNA levels of the related enzyme, butyryl-cholinesterase, nor of the high-affinity choline transporter. A γ-secretase inhibitor did not affect AChE transcript levels or enzyme activity in SN56 (APP695) or SH-SY5Y (APP695) cells, showing that regulation of AChE by APP does not require the generation of AICD or amyloid β peptide. Treatment of wild-type SN56 cells with siRNA targeting APP resulted in a significant up-regulation in AChE mRNA levels. Mutagenesis studies suggest that the observed transcriptional repression of AChE is mediated by the E1 region of APP, specifically its copper-binding domain, but not the C-terminal YENTPY motif. In conclusion, AChE is regulated in two neuronal cell lines by APP in a manner independent of the generation of sAPPα, sAPPβ, and AICD