Expression Of Amyloid-β Protein Precursor Research Articles

Mitochondrial Alterations in Neurons Derived from the Murine AppNL-F Knock-In Model of Alzheimer's Disease.

Alzheimer's disease (AD) research has relied on mouse models overexpressing human mutant A βPP; however, newer generation knock-in models allow for physiological expression of amyloid-β protein precursor (AβPP) containing familial AD mutations where murine AβPP is edited with a humanized amyloid-β (Aβ) sequence. The AppNL-F mouse model has shown substantial similarities to AD brains developing late onset cognitive impairment. In this study, we aimed to characterize mature primary cortical neurons derived from homozygous AppNL-F embryos, especially to identify early mitochondrial alterations in this model. Primary cultures of AppNL-F neurons kept in culture for 12-15 days were used to measure Aβ levels, secretase activity, mitochondrial functions, mitochondrial-ER contacts, synaptic function, and cell death. We detected higher levels of Aβ42 released from AppNL-F neurons as compared to wild-type neurons. AppNL-F neurons, also displayed an increased Aβ42/Aβ40 ratio, similar to adult AppNL-F mouse brain. Interestingly, we found an upregulation in mitochondrial oxygen consumption with concomitant downregulation in glycolytic reserve. Furthermore, AppNL-F neurons were more susceptible to cell death triggered by mitochondrial electron transport chain inhibition. Juxtaposition between ER and mitochondria was found to be substantially upregulated, which may account for upregulated mitochondrial-derived ATP production. However, anterograde mitochondrial movement was severely impaired in this model along with loss in synaptic vesicle protein and impairment in pre- and post-synaptic function. We show that widespread mitochondrial alterations can be detected in AppNL-F neurons in vitro, where amyloid plaque deposition does not occur, suggesting soluble and oligomeric Aβ-species being responsible for these alterations.

Exposure to Traffic-Generated Pollutants Exacerbates the Expression of Factors Associated with the Pathophysiology of Alzheimer's Disease in Aged C57BL/6 Wild-Type Mice.

Multiple studies report a strong correlation between traffic-generated air pollution-exposure and detrimental outcomes in the central nervous system (CNS), including Alzheimer's disease (AD). Incidence of AD is rapidly increasing and, worldwide, many live in regions where pollutants exceed regulatory standards. Thus, it is imperative to identify environmental pollutants that contribute to AD, and the mechanisms involved. We investigated the effects of mixed gasoline and diesel engine emissions (MVE) on the expression of factors involved in progression of AD in the hippocampus and cerebrum in a young versus aged mouse model. Young (2 months old) and aged (18 months old) male C57BL/6 mice were exposed to either MVE (300μg/m3 PM) or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-qPCR were used to quantify oxidative stress (8-OHdG) and expression of amyloid-β protein precursor (AβPP), β secretase (BACE1), amyloid-β (Aβ), aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP) 1B1, angiotensin-converting enzyme (ACE1), and angiotensin II type 1 (AT1) receptor in the cerebrum and hippocampus, in addition to cerebral microvascular tight junction (TJ) protein expression. We observed age-related increases in oxidative stress, AhR, CYP1B1, Aβ, BACE1, and AT1 receptor in the CA1 region of the hippocampus, and elevation of cerebral AβPP, AhR, and CYP1B1 mRNA, associated with decreased cerebral microvascular TJ protein claudin-5. MVE-exposure resulted in further promotion of oxidative stress, and significant increases in AhR, CYP1B1, BACE1, ACE1, and Aβ, compared to the young and aged FA-exposed mice. Such findings suggest that MVE-exposure exacerbates the expression of factors in the CNS associated with AD pathogenesis in aged populations.

Inhibitory effects of curcumin on H2O2-induced cell damage and APP expression and processing in SH-SY5Y cells transfected with APP gene with Swedish mutation.

Alzheimer's disease (AD) is a neurodegenerative disorder, and the pathological mechanism of the disease is still far to understand. According to the amyloid cascade hypothesis in AD, Amyloid-β (Aβ) is considered as a key substance that contributes AD development. Aβ is a β-cleaving product from Amyloid-β protein precursor (APP). Mutations of APP including APPKM670/671670NL (Swedish mutation) result in Aβ overproduction and the development of early-onset familial AD. Increase of oxidative stress and damage also occurs in early stage of AD. In this study, we used a SH-SY5Y cell line that stably expresses APP gene with Swedish mutation (SH-SY5Y-APPswe), and the inhibitory effects of curcumin on H2O2-induced cell damage and APP processing were investigated. Cells were treated with curcumin (0 ~ 5μM) for 4h before hydrogen peroxide (H2O2). Cell growth was detected with CCK-8 assay, and cell damage was determined through the evaluation of release of lactate dehydrogenase (LDH) from the cytosol to the culture medium and the morphological change of nucleus. The ability of mitochondrial stress and the depolarization of mitochondrial membrane potential were assayed through the measuring the oxygen consumption rate (OCR) and the green/red fluorescence ratio of JC-1 dye respectively. The protein levels of APP, sAPPα, sAPPβ, and BACE1 were analyzed with Western blot assay. Aβ production was measured with enzyme-linked immunosorbent assay (ELISA). The results indicated that curcumin inhibits H2O2-induced decrease of cell growth and cell damage. Curcumin attenuates H2O2-induced damage on the ability to mitochondrial oxidative phosphorylation and membrane potential. Curcumin inhibits H2O2-induced increase of APP cleavage through β-cleavage pathway and of intracellular Aβ production. These results imply that curcumin can be used to treat AD through inhibiting oxidative damage-induced APP β-cleavage and intracellular Aβ generation.

Knockout of Amyloid β Protein Precursor (APP) Expression Alters Synaptogenesis, Neurite Branching and Axonal Morphology of Hippocampal Neurons.

The function of the β-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

Dysregulation of Amyloid-β Protein Precursor, β-Secretase, Presenilin 1 and 2 Genes in the Rat Selectively Vulnerable CA1 Subfield of Hippocampus Following Transient Global Brain Ischemia.

The interaction between brain ischemia and Alzheimer’s disease (AD) has been intensively investigated recently. Nevertheless, we have not yet understood the nature and mechanisms of the ischemic episodes triggering the onset of AD and how they influence its slow progression. The assumed connection between brain ischemia and the accumulation of amyloid-β (Aβ) peptide awaits to be clearly explained. In our research, we employed a rat cardiac arrest model to study the changes in gene expression of amyloid-β protein precursor (AβPP) and its cleaving enzymes, β- and γ-secretases (including presenilins) in hippocampal CA1 sector, following transient 10-min global brain ischemia. The quantitative reverse-transcriptase PCR assay demonstrated that the expression of all above genes that contribute to Aβ peptide generation was dysregulated during 30 days in postischemic hippocampal CA1 area. It suggests that studied Aβ peptide generation-related genes can be involved in AβPP metabolism, following global brain ischemia and will be useful to identify the molecular mechanisms underpinning that cerebral ischemia might be an etiological cause of AD via dysregulation of AβPP and its cleaving enzymes, β- and γ-secretases genes, and subsequently, it may increase Aβ peptide production and promote the gradual and slow development of AD neuropathology. Our data demonstrate that brain ischemia activates delayed neuronal death in hippocampus in an AβPP-dependent manner, thus defining a new and important mode of ischemic cell death.

Upregulation of mitochondrial ferritin by proinflammatory cytokines: implications for a role in Alzheimer's disease.

Studies have shown an increased expression of mitochondrial ferritin (FtMt) and an antioxidant role for the protein in the brains of Alzheimer's disease (AD) patients. However, little information is available concerning the role of FtMt in other AD pathologies, including inflammation and amyloidogenesis. Therefore, we investigated the regulation and function of FtMt in inflammation and amyloidogenesis. FtMt protein expression was increased by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6), whereas FtMt mRNA levels were increased by TNF-α but not by IL-1β or IL-6 in IMR-32 cells. The transcription factor nuclear factor-κB (NF-κB) inhibitor, Bay 11-7082, suppressed this TNF-α-induced FtMt expression. FtMt overexpression increased NF-κB activity and translocation of p65 into the nucleus in HEK293 cells. Conversely, knockdown of FtMt attenuated TNF-α-induced NF-κB activity. Overexpression of FtMt inhibited TNF-α-induced apoptosis in the cell culture. FtMt overexpression reduced iron-mediated expression of amyloid-β protein precursor and decreased NF-κB-dependent increases in β- and γ-secretase, leading to decreased amyloid-β production. Our data provide new insights into the mechanism underlying the regulation of FtMt expression by proinflammatory cytokines and indicate further roles for FtMt in AD.

Identification of the Amyloid β-Protein Precursor and Cystatin C as Novel Epidermal Growth Factor Receptor Regulated Secretory Proteins in Nasopharyngeal Carcinoma by Proteomics

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.

X11 Proteins Regulate the Translocation of Amyloid β-Protein Precursor (APP) into Detergent-resistant Membrane and Suppress the Amyloidogenic Cleavage of APP by β-Site-cleaving Enzyme in Brain

X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid beta-protein precursor (APP) suppresses the generation of amyloid beta-protein (Abeta) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853-37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced beta-site cleavage of APP along with increased accumulation of Abeta in brain and increased colocalization of APP with beta-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its beta-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Abeta even if BACE activity did not increase in brain.

Inclusion-body myositis and Alzheimer disease

Inclusion-body myositis (IBM) is an age-related, slowly progressive degenerative disorder of the skeletal musculature with no effective treatment. Clinically, the disease is characterized by the atrophy of both proximal and distal muscle groups, leading to a gradually advancing weakness.1 The muscle fibers from IBM patients display characteristic vacuolizations and contain heterogeneous, filamentous inclusions.2 Although initially characterized as a form of polymyositis, the disease is only weakly responsive to corticosteroid treatment or other forms of immunomodulation and is now recognized as a distinct disease entity. Aspects of IBM pathology share intriguing similarities to that of Alzheimer disease (AD), the most common form of elderly dementia. The severe cognitive dysfunction in AD is brought about by massive neurodegenerative changes to the brain. The bulk of available evidence implicates a small protein fragment, the amyloid-β peptide (Aβ), as the major causative element in the neurodegenerative process.3,4 In the AD brain, Aβ is deposited extracellularly as amyloid, and is the major proteinaceous component of the neuritic plaques that can be found throughout the cortex. For over a decade, it has been known that Aβ—or larger protein fragments that contain it—also accumulate as part of the characteristic intracellular lesions of IBM.2,5,6 What is unknown is whether or not this is important for IBM pathogenesis. Strong evidence for a role of the amyloid β-protein precursor (APP), APP fragments, Aβ, or all three, in the pathogenesis of IBM comes from several sources.6,7 The appearance of Aβ-positive, noncongophilic deposits precedes vacuolization in IBM muscle fibers.8 Further, APP mRNA is increased in the disease state,9 suggesting that the over expression of the precursor protein may be sufficient to cause pathology. As expected, the in vitro over expression of APP in muscle cells leads to a range of degenerative changes resembling various facets …

Amyloid β protein precursor is involved in the growth of human colon carcinoma cellin vitro andin vivo

Amyloid beta protein precursor (APP) is a membrane-bound protein ubiquitously expressed in a variety of types of cells. However, its biological functions remain largely uncertain, particularly in non-neural cells and tumors. Our previous studies revealed that a secreted form of APP having a Kunitz-type inhibitor domain is a major serine proteinase inhibitor secreted by human colon carcinoma cells. In our study, we used an antisense RNA strategy to selectively inhibit the expression of APP in the human colon carcinoma cell line SW837. A vector capable of expressing an antisense mRNA complementary to 911 bases of the 5' end of APP mRNA was transfected into SW837 cells. After selection, 2 stably transfected antisense clones were obtained in which both the APP protein and mRNA were significantly suppressed. The proliferative potential and colony-forming efficiency of the antisense clones in vitro were markedly suppressed compared with the parent and mock-transfected clones. The addition of the conditioned medium of parent cells or purified secretory APP enabled these antisense effects to be overcome in vitro. The suppressed growth was also observed in vivo when the cells were injected subcutaneously into nude mice. Histologically, formation of tubular structures appeared to be suppressed in the antisense clones in vivo. These observations suggest potentially important roles of APP in cellular proliferation and differentiation of colon carcinoma cells.

Accumulation of amyloid β-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain

In aged rat brain, amyloid β-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and car☐y-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process.

Cellular specificity and regional distribution of amyloid β protein precursor alternative transcripts are unaltered in Alzheimer hippocampal formation

Studies comparing the expression of the amyloid β protein precursor (APP) gene in brains of patients with Alzheimer's disease (AD) and control individuals have resulted in contradictory findings indicative of selective reductions and relative increases of APP alternative transcripts in AD brain. It has been suggested that changes in APP expression in relation to AD neuropathology may represent highly specific and localized events involving only select populations of cells in particular brain regions. For example, reported AD-related alterations in ratios of APP alternative transcripts could be attributed to changes in expression of APP in specific neuronal subpopulations, and/or reactive astrocytes and/or microglia. To address this question, we have employed in situ hybridization using biotinylated oligonucleotide probes designed to localize specific APP mRNA transcripts in the hippocampal formation of AD patients and age-matched controls since this method allows a clear distinction of the classes of neurons and glia containing a particular message. Our findings can be summarized as follows: (1) APP695 and APP751 mRNAs are made primarily by pyramidal neurons in both AD and age-matched control hippocampal sections, (2) the cellular specificity and regional distribution of these messages are extremely similar not only with respect to each other, but also in AD and control hippocampal formation, (3) glial cells appear to contain only small or negligible amounts of APP message, and even in affected regions of AD brain displaying marked gliosis, reactive astrocytes do not contain increased amounts of APP RNA, and (4) APP695 and APP751 mRNAs were localized to both the soma and neuronal processes of neurons but dendritic staining for both messages appeared to be less pronounced in the AD cases.

Amyloid β-protein precursor (APP) of cultured cells: Secretory and non-secretory forms of APP

Overproduction or aberrant catabolism of the predicted amyloid β-protein precursor (APP) is suspected as the cause of amyloid deposition in Alzheimer's disease and Down's syndrome brains. For possible in vitro experiments of amyloid formation, we have examined the expression of APP in various cultured cells. We found two types of APP producing cell lines. PC12h (rat pheochromocytoma) and HL-60 (human acute promyelocytic leukemia) cells produce a secretory form that is released into the culture medium, while Bu-17 (human glioma) cells synthesize only a non-secretory form that accumulates at the cell surface. APP immunoreactivity on the latter cells was detected at the tips of cell processes or growth cones. These observations indicate that the nonsecretory form of APP may play a role in cell contact or adhesion.