Molecular misreading in non-neuronal cells.

+1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin(+1) protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too, could be affected by cellular derangements caused by molecular misreading.     

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.     

The Alzheimer amyloid precursor-related transcript lacking the beta/A4 sequence is specifically increased in Alzheimer's disease brain

The deposition of cerebrovascular and plaque amyloid in the CNS is a primary feature of Alzheimer's disease and aged Down's syndrome pathology. The localization of the Alzheimer amyloid protein precursor (APP) gene on chromosome 21, along with its overexpression in Down's syndrome brain compared with normal brain, suggests that alterations in APP gene expression may play a role in the development of the neuropathology common to the two diseases. In the present report, we demonstrate that a specific spliced form of mRNA that is transcribed from the APP gene and that lacks the beta/A4 sequence is elevated in the nucleus basalis, occipitotemporal cortex, and parahippocampal gyrus in Alzheimer's disease brain relative to controls. These results are based on combined data from RNA slot blot analysis, in situ hybridization, and polymerase chain reaction quantification of specific mRNAs taken directly from tissue sections.     

Beta-amyloid: Alzheimer's disease and brain beta-amyloidoses.

This review considers some aspects of the biochemistry of beta-amyloid, a protein which produces insoluble deposits in the brain. These deposits are a specific morphological feature of Alzheimer's disease, Down's syndrome, and senile dementia. Our contribution to the concept of a soluble form of beta-amyloid as of a normal human protein is presented.     

Frameshifted beta-amyloid precursor protein (APP+1) is a secretory protein,and the level of APP+1 in cerebrospinal fluid is linked to Alzheimer pathology

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1. Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP+1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP+1 level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) (p < 0.001), and the level of APP+1 in CSF was inversely correlated with the severity of the neuropathology. Moreover the earliest neuropathological changes are already reflected in a significant decrease of the APP+1 level in CSF. These data show that APP+1 is normally secreted by neurons, preventing intra-neuronal accumulation of APP+1 in brains of non-demented controls without neurofibrillary pathology.     

Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.     

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.     

Detection of soluble forms of the beta-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.     

Alzheimer's disease untangled.

The last year has seen major advances in the study of Alzheimer's disease (AD). Four mutations involving amino acid substitutions in exons 16 and 17 of the amyloid precursor protein (APP) gene, have been identified which co-segregate with the disease in some families multiply affected by early onset Alzheimer's disease. These mutations are strongly suggestive of a causative role for the amyloid precursor protein in Alzheimer's disease. Despite their rarity, these mutations are important because they represent the first known cause of Alzheimer's disease. Processing of APP must be central to the pathogenesis of the disease although the precise effects of these amino acid substitutions are not understood. Work is now being undertaken to characterise the processing pathways of APP and to identify other causes of AD. The development of models of AD using the APP mutations offers the possibility of identifying drug targets and developing more effective treatments than are presently available.     

SEL-10 interacts with presenilin 1, facilitates its ubiquitination,and alters A-beta peptide production

Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.     

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.     

A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of Alzheimer's disease.

Amyloid deposits in the brains of patients with Alzheimer's disease (AD) contain a protein (beta A4) which is abnormally cleaved from a larger transmembrane precursor protein (APP). APP is believed to be normally released from membranes by the action of a protease referred to as APP secretase. Amyloid deposits have also been shown to contain the enzyme acetylcholinesterase (AChE). In this study, a protease activity associated with AChE was found to possess APP secretase activity, stimulating the release of a soluble 100K form of APP from HeLa cells transfected with an APP cDNA. The AChE-associated protease was strongly and specifically inhibited by soluble APP (10 nM) isolated from human brain. The AChE-associated protease cleaved a synthetic beta A4 peptide at the predicted cleavage site. As AChE is decreased in AD, a deficiency of its associated protease might explain why APP is abnormally processed in AD.     

Amyloid precursor protein family-induced neuronal death is mediated by impairment of the neuroprotective calcium/calmodulin protein kinase IV-dependent signaling pathway

The aberrant metabolism of beta-amyloid precursor protein (APP) and the progressive deposition of its derived fragment beta-amyloid peptide are early and constant pathological hallmarks of Alzheimer's disease. Because APP is able to function as a cell surface receptor, we investigated here whether a disruption of the normal function of APP may contribute to the pathogenic mechanisms in Alzheimer's disease. To this aim, we generated a specific chicken polyclonal antibody directed against the extracellular domain of APP, which is common with the beta-amyloid precursor-like protein type 2. Exposure of cultured cortical neurons to this antibody (APP-Ab) induced cell death preceded by neurite degeneration, oxidative stress, and nuclear condensation. Interestingly, caspase-3-like protease was not activated in this neurotoxic action suggesting a different mode of cell death than classical apoptosis. Further analysis of the molecular mechanisms revealed a calpain- and calcineurin-dependent proteolysis of the neuroprotective calcium/calmodulin-dependent protein kinase IV and its nuclear target protein cAMP responsive element binding protein. These effects were abolished by the G protein inhibitor pertussis toxin, strongly suggesting that APP binding operates via a GTPase-dependent pathway to cause neuronal death.