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

The toxic role of amyloid ß peptides in Alzheimer's disease is well documented. Their generation is via sequential ß- and γ-secretase cleavage of the membrane-bound amyloid precursor protein (APP). Other APP metabolites include the soluble ectodomains sAPPα and sAPPß 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 ß 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, sAPPß, and AICD.

The Amyloid Precursor Protein Plays Differential Roles in the UVA Resistance and Proliferation of Human Retinal Pigment Epithelial Cells.

BACKGROUND: Age-related macular degeneration (AMD) can be characterised by degeneration of retinal pigment epithelial (RPE) cells and the accumulation, in retinal drusen deposits, of amyloid beta-peptides proteolytically derived, by secretases, from the amyloid precursor protein (APP). Ultraviolet (UV) light exposure is a risk factor for the development of AMD. OBJECTIVES: In the current study, we investigated whether APP and/or its proteolysis are linked to the UVA resistance or proliferation of ARPE-19 human RPE cells. METHODS: Cell viability was determined, following UVA exposure, with prior small interfering RNA-mediated APP depletion or secretase inhibitor treatments. APP levels/proteolysis were analysed by immunoblotting. Cells were also grown in the presence/absence of secretase inhibitors to assess their effects on longer-term culture growth. Finally, the effects of APP proteolytic fragments on ARPE-19 cell proliferation were monitored following co-culture with human embryonic kidney cells stably over-expressing these fragments. RESULTS: Endogenous APP was depleted following UVA irradiation and ß-secretase, but not α- secretase, the processing of the protein was reduced. Experimental APP depletion or γ-secretase (but not α- or ß-secretase) inhibition ablated the detrimental effect of UVA on cell viability. In contrast, α-secretase, and possibly γ-secretase but not ß-secretase activity, appeared to promote the longerterm proliferation of ARPE-19 cells in the absence of UVA irradiation. CONCLUSION: There are clear but differential links between APP expression/proteolysis and the proliferation and UVA resistance of ARPE-19 cells indicating that the protein should be investigated further in relation to the identification of possible drug targets for the treatment of AMD.

The orphan drug dichloroacetate reduces amyloid beta-peptide production whilst promoting non-amyloidogenic proteolysis of the amyloid precursor protein.

The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by ß- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting ß-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the ß-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, ß- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.

Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17, but is not a lipid-raft-associated event.

Notch signalling is an evolutionarily conserved pathway involved in cell-fate specification. The initiating event in this pathway is the binding of a Notch receptor to a DSL (Delta/Serrate/Lag-2) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalysed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch-binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signalling. In the present study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a GPI (glycosylphosphatidylinositol) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid-raft-independent manner, and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding.

Gene therapy-mediated enhancement of protective protein expression for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the leading form of dementia but lacks curative treatments. Current understanding of AD aetiology attributes the development of the disease to the misfolding of two proteins; amyloid-ß (Aß) and hyperphosphorylated tau, with their pathological accumulation leading to concomitant oxidative stress, neuroinflammation, and neuronal death. These processes are regulated at multiple levels to maintain homeostasis and avert disease. However, many of the relevant regulatory proteins appear to be downregulated in the AD-afflicted brain. Enhancement/restoration of these 'protective' proteins, therefore, represents an attractive therapeutic avenue. Gene therapy is a desirable means of achieving this because it is not associated with the side-effects linked to systemic protein administration, and sustained protein expression virtually eliminates compliance issues. The current article represents a focused and succinct review of the better established 'protective' protein targets for gene therapy enhancement/restoration rather than being designed as an exhaustive review incorporating less validated protein subjects. In addition, we will discuss how the risks associated with uncontrolled or irreversible gene expression might be mitigated through combining neuronal-specific promoters, inducible expression systems and localised injections. Whilst many of the gene therapy targets reviewed herein are yet to enter clinical trials, preclinical testing has thus far demonstrated encouraging potential for the gene therapy-based treatment of AD.

Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. PrP(C) is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrP(C) is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrP(C). These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrP(C) and that ADAM9 exerts its effect on PrP(C) shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly(228) and Arg(229), three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein beta-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrP(C) shedding or activation of both PrP(C) and PrP(Sc) shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrP(Sc). Collectively, these data indicate that although PrP(C) can be shed through the action of ADAM family members, modulation of PrP(C) or PrP(Sc) ectodomain shedding does not regulate prion conversion.

Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein.

Proteolytic processing of the amyloid precursor protein (APP) by beta-secretase, beta-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid beta (Abeta) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP(C)), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrP(C) are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP(C) in the proteolytic processing of APP. Cellular overexpression of PrP(C) inhibited the beta-secretase cleavage of APP and reduced Abeta formation. Conversely, depletion of PrP(C) in mouse N2a cells by siRNA led to an increase in Abeta peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Abeta levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the beta-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP(C) on the beta-secretase cleavage of APP required the localization of PrP(C) to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP(C) via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Abeta is regulated by PrP(C) and may have implications for both Alzheimer's and prion diseases.

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.

Herpes simplex virus interferes with amyloid precursor protein processing.

BACKGROUND: The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-epsilon4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39-43 amino acid protein--beta amyloid (Abeta)--within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Abeta or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Abeta; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting. RESULTS: We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels--as might be expected--yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide CONCLUSION: Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD.