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PubMed2000

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells.

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.

ADAM Proteins↗
PubMed1997

Effects of cadmium, copper, and zinc and beta APP processing and turnover in COS-7 and PC12 cells. Relationship to Alzheimer disease pathology.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Amyloid Precursor Protein Secretases↗
PubMed2003

Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5.

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. One of the protein kinases known to phosphorylate APP(Thr668) is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP(Thr668) phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP(Thr668) phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP(Thr668) phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.

Amyloid beta-Protein Precursor↗
PubMed1996

A new quantitative solution hybridisation-RNase protection assay for APP and APLP2 mRNA.

Amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) are members of a multigene family of proteins implicated in the pathogenesis of Alzheimer's disease. We describe the development of an RNA-RNA solution hybridisation-RNase protection assay to quantify APP mRNA. APP mRNA splice forms containing the Kunitz-type protease inhibitor (KPI) insert, and APLP2 mRNA in total nucleic acid extracts from a range of tissue types. Solution hybridisation-RNase protection assay enables absolute quantification of target mRNA, by conversion of the hybridisation signal to pg mRNA using a standard curve. The assay is sensitive, capable of detecting 1 pg target mRNA, and reproducible, with an inter-assay variability of less than 10% and an intra-assay variability of 3-4%. We quantified APP and APLP2 mRNA in cell lines and post-mortem human brain tissue samples. To test whether we could detect physiological differences in APP mRNA levels, a fibroblast cell line with a paternal chromosome 21 deletion of the region including the APP gene was analysed and found to express half as much APP mRNA as control fibroblasts. In addition, a reversible, approx. 30% increase in APP mRNA levels was detected in human lymphoblastoid cell lines following heat shock, a physical stimulus previously shown to increase APP expression. Regional differences in the expression of APP and APLP2 were seen in human post-mortem cerebral cortex and cerebellum. Levels of APP and APLP2 mRNA were highest in the temporal cortex, slightly lower in frontal and occipital cortices, and lowest in the cerebellum. The highest proportion of KPI-containing APP was seen in the frontal and temporal cortices. The ratio of APP:APLP2 mRNA was 1:0.3 in the cortical tissue and 1:0.8 in the cerebellum. In conclusion, quantitative solution hybridisation-RNase protection assay of total APP. APP KPI and APLP2 mRNA provides a new tool to improve the resolution of studies of potentially subtle alterations in the expression of these genes in both cell culture model systems and Alzheimer's disease post-mortem human brain tissue.

Amyloid beta-Protein Precursor↗
PubMed2000

Regulation of amyloid precursor protein (APP) secretion by protein kinase calpha in human ntera 2 neurons (NT2N).

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic APP products. Protein kinase C (PKC) has been shown to regulate APPs secretion, and PKCalpha and PKCepsilon have been implicated in APPs secretion in fibroblasts. This study examined the PKC isoform involved in regulated APPs secretion in human NT2N neurons and in CHO cells stably expressing APP(695). Inhibition of PMA-induced APPs secretion with the PKC inhibitors Calphostin C and GF109203X demonstrated that PKC is involved in PMA-regulated APPs secretion in NT2N cells. The specific PKC isoforms present in NT2N and CHO695 cells were identified, and PKCalpha and PKCepsilon were found to translocate from cytosol to membranes in NT2N and CHO695 cells. Translocation of PKC to the membrane allows for activation of the enzyme, as well as for positioning of the enzyme close to its substrate. Long-term PMA treatment led to complete downregulation of PKCalpha in NT2N cells and to downregulation of PKCalpha and PKCepsilon in CHO695 cells. PKCalpha downregulation in the NT2N cells resulted in loss of PMA-regulated APPs secretion and a substantial reduction in constitutive APPs secretion. Downregulation of PKCalpha and PKCepsilon in CHO695 cells resulted in loss of PMA-regulated APPs secretion; however, constitutive APPs secretion was unaffected. These findings suggest that PKCalpha is involved in PMA-regulated APPs secretion in NT2N cells and PKCalpha and/or PKCepsilon is involved in PMA-regulated APPs secretion in CHO695 cells.

Amyloid beta-Protein Precursor↗
PubMed2000

Regulation of APP synthesis and secretion by neuroimmunophilin ligands and cyclooxygenase inhibitors.

We and others previously showed that both the synthesis of the amyloid precursor protein (APP) and its processing (i.e., to amyloidogenic A beta peptides; soluble nonamyloidogenic APPs; and other APP fragments) are regulated by neurotransmitters. Transmitters that elevate cellular cAMP levels (like norepinephrine and prostaglandins, which act on beta-adrenergic receptors and prostaglandin E2 receptors respectively) enhance APP synthesis and the formation of amyloidogenic APP holoprotein. Transmitters that stimulate phosphatidylinositol hydrolysis (by activating muscarinic m1 or m3 receptors, serotoninergic 5HT2a or 5HT2c receptors, or metabotropic glutamate receptors of subtypes 1 or 5) increase the conversion of APP to soluble APPs, and decrease the formation of A beta. These findings suggest that drugs that regulate the activity of neurotransmitter receptors might be useful in preventing the excessive formation of A beta or other amyloid precursors in Alzheimer's disease. We now show that neuroimmunophilin ligands (like cyclosporin A or FK-506) and nonsteroidal antiinflammatory agents (NSAIDs), including cyclooxygenase (COX)-2 inhibitors, can also prevent APP overexpression and the overproduction of amyloidogenic peptides. We observe that the enhancement of APP overexpression by prostaglandin E2 is inhibited by neuroimmunophilin ligands like cyclosporin A or FK-506 (tacrolimus). We also find that the NSAIDs, which reduce prostaglandin synthesis by inhibiting COX-1 and -2 enzymes, might also be expected to lower APP levels. Our present data confirm that these drugs, as well as drugs that selectively inhibit COX-2, reduce the levels of amyloidogenic APP holoprotein in cultured neurons or in cultured astrocytes. We previously showed that elevations in cAMP, perhaps generated in response to prostaglandins, can suppress APPs secretion. The NSAIDs and COX inhibitors also increased levels of soluble APPs in the media of cultured astrocytes and neurons, perhaps acting by inhibition of prostaglandin production. Since APP holoprotein can be amyloidogenic, while APPs may be neurotrophic, our findings suggest that some neuroimmunophilin ligands, NSAIDs and COX-2 inhibitors might suppress amyloid formation and enhance neuronal regeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed2000

Platelet APP isoform ratios correlate with declining cognition in AD.

BACKGROUND: Platelets and neurons both contain large quantities of two carboxyl-truncated 120 to 130 and 110 kDa Alzheimer amyloid precursor proteins (APPs). Platelets taken from patients with AD have been reported to contain a reduced ratio of these APPs. OBJECTIVE: To further study the AD specificity of reduced platelet APP ratios and to determine whether, after 3 years, cognitive losses in AD are accompanied by similarly reduced platelet APP ratios. METHODS: To test the AD specificity of reduced platelet APP ratios, we quantitated these APPs in eight patients with PD and six patients with hemorrhagic stroke (HS). To determine whether further cognitive losses correlate with platelet APP ratio reductions in patients with AD, the authors re-examined platelet APPs and Mini-Mental State Examination (MMSE) scores of 10 patients with AD and 11 controls, who were tested 3 years ago. APP ratios were determined by the average of six assays using Western blotting with m22C11 monoclonal antibody, enhanced chemoluminescence, and digital scanning of autoradiographs. RESULTS: APP ratios were normal in the patients with PD and HS, further supporting the AD specificity of this assay. After 3 years, the MMSE scores and APP ratios of our control subjects changed by <4%. However, the average MMSE scores of our patients with AD declined from 16.4 to 8.3, and their average 120 to 130/110 kDa APP ratios declined from 5.8 to 3.6. The difference between AD and control APP ratios, with no overlap, is significant and the correlation between the 3-year decline in AD MMSE scores and reduced APP ratios (r = 0.69) was significant. CONCLUSIONS: Although the number of subjects analyzed was limited, reduced platelet APP ratios appear to be a specific biological marker of AD and a biological index of the severity of cognitive loss in AD.

Aged↗
PubMed1995

Particulate forms of APP in the extracellular milieu of cultured cells.

The principle externalized forms of amyloid precursor protein (APP) are soluble and well-characterized, but some evidence has suggested the additional presence of externalized APP in a nonsoluble form. To further assess this possibility, the current study has applied high resolution microscopy protocols in addition to immunoprecipitation to characterize externalized APP in three commonly used cell culture models (SH-SY5Y human neuroblastoma cells, fetal rat brain cells, and HEK 293 human embryonic kidney cells). Confocal immunofluorescence microscopy, using an antiserum against the c-terminal domain of APP, showed typical cell-associated APP, but hot spots of APP also were evident in cell-free areas, apparently associated with the culture substrata. These hot-spots were examined for evidence of cellular deterioration by whole mount transmission electron microscopy. Neither cell debris nor disrupted cells were present. Instead, the hot spots of substratum-bound APP comprised discrete microparticles, approximately 50-100 nm across. These microparticles also could be found near cells and in some cases were attached to cell surface fibrils. Substratum-bound APP also could be found clustered within the extracellular matrix made by primary cell cultures. Occurrence of APP in extracellular microparticles was verified by centrifugation-immunoprecipitation analysis of media conditioned by APP-transfected cells. Radiolabeling data showed that particulate APP was from metabolically active cells. Metabolic labeling of particle-associated APP, as well as the absence of cellular debris near the APP-containing particles, suggests that the occurrence of nonsoluble APP in the extracellular milieu derives from a physiologically active process.

Amyloid beta-Protein Precursor↗
PubMed1996

Quantification of APP and APLP2 mRNA in APOE genotyped Alzheimer's disease brains.

Amyloid precursor protein (APP) is metabolised to produce A beta, a peptide found aggregated in Alzheimer's disease neuritic plaques. APP is a member of a multigene protein family which includes amyloid precursor-like protein 2 (APLP2). Since A beta accumulation can be triggered by factors acting up- or downstream of APP processing, we investigated whether APP mRNA expression was altered in Alzheimer's disease post-mortem cerebral cortex. In addition, we characterised cortical APLP2 mRNA levels. Quantitative RNA-RNA solution hybridisation-RNase protection was used to assay total APP. APP containing the Kunitz-type protease inhibitor (KPI) insert and APLP2 mRNA in mid-temporal and superior frontal cortices from apolipoprotein E-genotyped subjects with Alzheimer's disease, other neurological diseases and non-demented controls. Approximately 3 times more APP than APLP2 mRNA was detected and about 70% of total APP mRNA contained the KPI insert in the control subjects. Total APP and APLP2 mRNA levels were significantly reduced in Alzheimer's disease mid-temporal, but not superior frontal cortex, suggesting that regional reductions in these mRNA correlate with severity of disease pathology. A small significant increase in the proportion of APP KPI mRNA was seen in both cortical regions in Alzheimer's disease. Apolipoprotein E genotype did not influence cortical levels of total APP, APP KPI or APLP2 mRNA. Alzheimer's disease-related increases in tissue DNA content were seen in both regions studied, while tissue RNA levels were reduced in the positive disease controls. In summary, these results indicate that Alzheimer's disease is not associated with over-expression of either APP or APLP2 mRNA. Our findings reveal a disease-associated increase in the proportion of APP KPI-containing isoforms, and further investigation should clarify whether this predisposes affected individuals to A beta production and aggregation, or reflects later events such as gliosis and neuronal cell death.

Aged↗
PubMed2026

Smartphone Apps for Preventing Adolescent Health Problems Among Health Care Professionals: Systematic Search and Quality Assessment.

BACKGROUND: Health care professionals must consider multiple dimensions of prevention when consulting with adolescents. Identifying risky behaviors early in adolescence is crucial for reducing both morbidity and mortality. General practitioners are increasingly eager to incorporate digital tools for prevention into their consultations with adolescents; however, the relevance and clinical validity of these digital tools are not always established or well-known. Consequently, primary care professionals require guidance and support in selecting relevant mobile health (mHealth) tools. OBJECTIVE: The aim of this study is to identify relevant and useful digital apps to help primary care professionals detect at-risk adolescents across all recommended areas of prevention: orthopedics, mental health, substance abuse, risk behaviors, sexual health, vaccinations, social relationships, and nutrition. METHODS: A systematic review of smartphone apps, with an analysis of content quality, was carried out by 4 researchers using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) checklist. The App Store and Google Play Store platforms were surveyed. The inclusion criteria were as follows: free of charge, date of last update, availability in French or English, relevance of the preventive approach to adolescents, and scientific validation. Four health care professionals assessed the apps: 2 selected the apps relevant to health care professionals, then 3 analyzed these apps using the French version of the Mobile App Rating Scale (MARS-F). Intraclass correlation coefficient, model (2,1) (2-way random effects, absolute agreement, single measures); standard error of measurement; and mean absolute error were also calculated. RESULTS: A total of 976 apps were identified, 49 of which had disappeared from the platforms prior to analysis. Nine apps were retained. Seven (0.72%) were included after evaluation using the MARS-F: 2 on mental health and 5 on sexual health (including 3 on contraception only). The mean MARS-F interrater score ranged from 2.5/5 to 3.8/5. The global MARS-F score demonstrated a pooled SD of 0.60 and an intraclass correlation coefficient (2,1) of 0.0003, resulting in a calculated standard error of measurement of 0.60. The average discrepancy between raters was a mean absolute error of 0.53. CONCLUSIONS: No similar studies have been identified in the literature that specifically focus on mobile apps designed to support health care professionals in delivering preventive care to adolescents. Of the 8 areas of prevention identified as relevant for adolescents, only 3 are addressed by the apps validated through our methodology (5 focus on sexual health). Consequently, current apps are insufficient to support health care professionals in their overall preventive work with adolescents. Such a review should be conducted systematically prior to the development of any new tool to prevent duplication and channel creative efforts toward truly innovative digital solutions. Furthermore, a thorough analysis of relevant, recommended websites is essential, as these resources complement the use of mobile apps designed for health care professionals.

Humans↗
PubMed1997

Interaction between Alzheimer's disease beta A4 precursor protein (APP) and the extracellular matrix: evidence for the participation of heparan sulfate proteoglycans.

The interaction between the Alzheimer amyloid precursor protein (APP) and an intact extracellular matrix (ECM), matrigel, obtained from Engelbreth-Holm-Swarm tumors was evaluated. Based on quantitative analyses of the binding data obtained from solid phase binding assays, two binding sites on the ECM were identified for [125I]-APP (with apparent Kd1 of 1.0 x 10(-11) M and Kd2 of 1.6 x 10(-9) M respectively). Over 70% of [125I]-APP was displaced by heparin and N-desulfated heparin but not by chondroitin sulfate. Pretreatment of matrigel with heparitinase decreased the binding of [125I]-APP by 80%. beta-amyloid peptides (residues 1-40, 1-28, and 1-16) containing a heparin binding domain also displaced 80% of bound [125I]-APP, which was totally displaced by intact APP. The binding of [125I]-APP to matrigel increased by 210% with a decrease in the pH. These observations suggest that [125I]-APP interacts mainly with heparan sulfate proteoglycan present in the ECM. The binding of [125I]-APP to individual ECM components was also analyzed. [125I]-APP was found to bind laminin and collagen type IV but not fibronectin. However, when these ECM constituents were combined, the extent of APP-binding decreased significantly, to levels comparable to those obtained with intact matrigel, suggesting that multiple interactions may occur between ECM constituents and [125I]-APP. The results are discussed in terms of APP function and amyloidogenesis.

Alzheimer Disease↗
PubMed1996

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma.

Thyroid epithelial cells have been shown to have a high APP expression and to produce large amounts of its metabolic derivatives, namely secreted APPs and a potentially amyloidogenic 41-kDa C-terminal fragment. It was the aim of the present study to analyze how APP production and metabolism were regulated in human thyroid cells. The effects of three cytokines, interferon gamma (IFN gamma), interleukin 1beta (IL-1 beta) and transforming growth factor (TGF) beta, were investigated. Cell extracts and supernatants were studied by immunoblotting using specific N- and C-terminal APP antibodies. Quantification was performed by densitometric scanning. We demonstrate that IFN gamma has a strong suppressive effect on the production and metabolism of APP. From a concentration of 30 U/ml upwards it reduces the cellular APP content, decreases the amounts of secreted APPs and inhibits the generation of the 41-kDa amyloidogenic APP fragment. In contrast, IL-1 beta has a stimulatory influence on the generation of the amyloidogenic 41-kDa APP metabolite, but does not affect the cellular holoprotein or APPs. TGFbeta has no significant effect on APP. Our results demonstrate that cytokines can regulate APP production and metabolism in thyroid cells. IFN gamma is the first naturally occurring agent described to inhibit the generation of amyloidogenic APP fragments. It may be of relevance in preventing amyloid deposition during inflammatory processes in the thyroid gland, but may exert a similar protective effect in other non-neuronal and neuronal tissues.

Adult↗
PubMed2000

Antibodies to the C-terminus of the beta-amyloid precursor protein (APP): a site specific marker for the detection of traumatic axonal injury.

Antibodies to the amyloid precursor protein (APP) are commonly used to detect traumatic axonal injury (TAI). Carried by fast anterograde axoplasmic transport, APP will pool at regions of impaired transport associated with TAI. Based primarily upon commercial antibody availability, previous studies have targeted the N-terminus of APP, which, with respect to antigen detection, is suboptimally located within anterogradely transported vesicles. Recently, antibodies to the APP C-terminus, located on the external surface of anterogradely transported vesicles, have become available, allowing for the exploration of their utility in detecting TAI. To this end, rats were subjected to an impact acceleration injury, surviving 30 min to 24 h post-injury. They were then perfused, their brains sectioned and prepared for dual label immunofluorescent microscopy, single label bright field microscopy, and electron microscopy (EM). Antibodies to the APP C-terminus yielded the ready detection of intensely labeled TAI with significantly reduced diffuse background staining in comparison to antibodies to the APP N-terminus in both dual label immunofluorescent and single label bright-field approaches. EM examination of antibodies to the APP C-terminus in TAI revealed intense labeling of pooled intra-axonal vesicular profiles, confirming the anterogradely transported vesicular source of the APP seen in TAI. Interestingly, in addition to providing a technically superior approach and new detailed information on the subcellular localization of APP, antibodies to the APP C-terminus also proved more cost effective. Immunofluorescent studies of APP C-terminus immunoreactivity involved 1/3 the cost of targeting the N-terminus, while bright field APP C-terminus studies were performed for 1/20 the cost.

Amyloid beta-Protein Precursor↗
PubMed2000

Endogenous APP derivatives oppositely modulate apoptosis through an autocrine loop.

We have recently shown that, in rat cerebellar granule cells, apoptosis triggered by KCl deprivation is associated with an amyloidogenic shift in the processing of amyloid precursor protein (APP) resulting in an increase of amyloid beta-protein (A beta) secretion. To further investigate this issue we studied the relationship between secretion of APP metabolites (A beta, APPs) and neuronal degeneration. We postulated that the endogenous products of the APP metabolism may modulate neuronal survival by an autocrine loop. Treatment of cerebellar granule cells with various antibodies raised against different epitopes of APPs and A beta oppositely modulates low potassium apoptotic cell death. Antibodies specific for the N-terminal of A beta (4G8, 6E10, R3659) increased neuronal survival by 30% over controls. On the contrary, treatment of cultures undergoing apoptosis with the monoclonal antibody 22C11 directed against the APP N-terminus reduced neuronal survival by 53%, suggesting that endogenous alpha-APPs contribute to neuronal survival. Moreover low KCl culture medium, conditioned by cerebellar granule cells, attenuated the apoptotic process. This anti-apoptotic effect was abolished by removal of APPs from the conditioned medium. Western blotting of APPs removed from the conditioned medium confirmed the presence of alpha-APPs. These data indicate that APP cleavage products oppositely modulate neuronal survival through an autocrine loop and further strengthen an Alzheimer's disease pathogenetic scheme based on altered metabolism of APP.

Amyloid beta-Protein Precursor↗
PubMed1996

Molecular physiology, biochemistry, and pharmacology of Alzheimer's amyloid precursor protein (APP).

The function of APP is not yet known in detail but growing evidence exists that APP may mediate cell interactions with the cell surface or soluble glycoproteins and defense mechanisms in the CNS involving the immune system. We describe here the finding that almost all CD4+ lymphocytes and the majority of CD8+ lymphocytes were positive for A beta and the antibodies against A beta or APP did not inhibit the [3H]-thymidine uptake of mitogen-treated lymphocytes significantly. There were no differences in the A beta immunoreactivity on the cell surface of lymphocytes between Alzheimer's disease (AD) and control samples. Excessive amyloidogenic pathway of APP processing may be the final common pathway involved in the pathogenesis of AD. Thus, the identification of proteases or factors leading to aberrant proteolysis which process APP to yield a variety of potentially amyloidogenic fragments would promise pharmacological targets to develop anti-AD drugs. In attempts to define the proteases or factors which alter the balance between nonamyloidogenic and amyloidogenic processing pathways, our study indicates that thrombin or acetylcholinesterase(AChE)-associated protease may be involved in the amyloidogenic processing pathway of APP in vivo to generate amyloidogenic intermediates linked to amyloid deposition. Highly specific and dose-dependent direct modulation of APP processing by biologically available metal ions including Ca2+, Zn2+, Fe2+/Fe3+ and Al3+ suggest the disrupted metal homeostasis as factors leading to overaccumulation of APP and subsequent aberrant proteolysis utilizing excessive amyloidogenic processing pathway. There is mounting evidence that at least some of the neurotoxicity associated with AD is due to fragments from APP. Most research has focused on the toxic effect and the ion channel activity of A beta in causation of the disease. The possible role of other cleaved products of APP is less clear. We investigated the channel-forming ability of various products of APP when applied to Xenopus oocytes and their neurotoxicity in vitro. CT105 peptide was found to be exceedingly potent at 500 nM concentration in forming nonselective ion channels during application from either outside or inside the oocyte and more toxic than either of the A beta fragments, A beta 25-35, or A beta 1-40. Taken together, these results suggest the possible involvement of CT peptide in inducing the neurotoxicity characteristic of AD through the direct damage on the cell membrane. Therefore, we hypothesize that amyloidogenic CT may make nonselective ion channels or pores in the membrane and may cause neuronal death in the early stage of AD and then further metabolized to more stable and less toxic A beta which may be finally deposited in the brain where it could inflict further toxicity to neurons. Here we report successful inhibition of APP gene expression by antisense oligodeoxynucleotides at the mRNA or the protein level in in vitro and cell culture systems.

Alzheimer Disease↗
PubMed2002

Regulation of APP-dependent transcription complexes by Mint/X11s: differential functions of Mint isoforms.

Mints/X11s are neuron-specific (Mints 1 and 2) and ubiquitous (Mint 3) adaptor proteins composed of isoform-specific N-terminal sequences and common C-terminal phosphotyrosine-binding (PTB) and PDZ domains. We now show that all three Mints bind to the cytoplasmic tail of amyloid-beta precursor protein (APP) and presenilins and strongly increase the levels of cellular APP in transfected cells. Immunocytochemistry revealed that in neurons, Mints 1 and 2 were colocalized with APP in the trans-Golgi network, with lower levels throughout the cell body and neurites. Using an APP-dependent transactivation assay that uses a fusion protein of APP coupled to the potent transcription factor Gal4/VP16, we examined the effects of Mints on the proteolytic processing and putative transcriptional function of APP. Although all Mints were biochemically similar, only Mints 1 and 2 but not Mint 3 strongly inhibited transactivation by APP-Gal4/VP16. Inhibition was enhanced by a mutation of the first PDZ domain and by deletion of the PDZ domains or the N-terminal sequences but abolished by inactivation of the PTB- and PDZ domains. Mint 1 also inhibited transactivation by the "precleaved" cytoplasmic tail of APP fused to Gal4/VP16, whereas Fe65 (which binds to APP as strongly as Mints) enhanced transactivation. Our data suggest that Mints 1 and 2 but not Mint 3 have a specific effect on APP function that cannot be explained simply by their interaction with presenilins and occurs at least partly after cleavage of APP. In view of their biochemical similarity, the functional differences among Mints are unexpected, suggesting that Mints 1 and 2 have a brain-specific function related to APP that is not executed by the ubiquitous Mint 3.

Adaptor Proteins, Signal Transducing↗
PubMed1993

APP+ T lymphocytes selectively sorted to endomysial tubes in polymyositis displace NCAM-expressing muscle fibers.

The characteristic pathogenic feature of polymyositis (PM) is muscle invasion by T lymphocytes penetrating the basal lamina and displacing the sarcolemma of normal muscle fibers (T cell invasion of endomysial tubes). Active forward movement of these T cells is indicated by cell extensions interdigitating with the muscle fiber surface. Here we describe for the first time high abundance of Alzheimer amyloid protein precursor (APP) in invasive T cells contacting the border of muscle fibers in PM. These are the sites of muscle fiber displacement. The percentage of APP+ T cells at these sites is significantly higher than in other neuromuscular disorders with inflammatory infiltrates suggesting a specific pathogenic function of these cells in PM. By using a new multiparameter immunofluorescence imaging procedure and confocal laser scanning microscopy, we show that APP+ T cells in PM are invasive front cells that penetrate the basal lamina of the endomysial tube and displace the muscle fiber. Mononuclear cells behind the invasive front are negative for APP or show much lower APP levels. Front T cells either express the CD8-CD4+APP+ or CD8+CD4-APP+ phenotypes, or are CD4+CD8+APP+ T cell chimeras. The highest APP concentration is found at the tip of T cell extensions interdigitating with the fiber surface. Although normal by morphological criteria, the same fibers show intense staining for the regeneration marker NCAM. This reactivity is highest at sites contacted by the APP+ T cells. The findings indicate that APP is specifically upregulated in T cells displacing muscle fibers in PM and suggest that NCAM, which may be abnormally regulated in these fibers, is a candidate molecule for interaction with APP.

Amyloid beta-Protein Precursor↗
PubMed1994

APP gene family: unique age-associated changes in splicing of Alzheimer's betaA4-amyloid protein precursor.

The betaA4-amyloid protein precursor (APP) is a transmembrane glycoprotein that is the source of the characteristic betaA4-amyloid deposits of Alzheimer brains. It exists in eight isoforms generated by alternative splicing of exons 7, 8 and 15, of which the L-APP mRNAs lacking exon 15 are significantly expressed in non-neuronal cells and tissues, but not in neurones. Recently, it was shown that APP is a member of a multigene family of which the amyloid precursor-like protein 2 (APLP2) is the nearest relative. Analysis of APLP2 expression revealed regulated alternative splicing of the Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a non-homologous insert of 12 amino acids on the NH2-terminal side of the transmembrane domain. While expression of the KPI encoding exon of APLP2 is abundant in neurones and thus differs from APP, L-APLP2 mRNA isoforms lacking the latter, non-homologous insert show a tissue-specific expression pattern similar to that of exon 15 of APP. Comparison of alternatively spliced APP and APLP2 mRNA isoforms in rat brain regions from early post-natal and adult rats revealed significantly higher relative amounts of KPI-encoding APP isoforms in the adult rat brain and an even more pronounced augmentation of L-APP mRNAs. Both effects were not observed for APLP2. This indicates an APP-specific age-associated regulation pattern within the APP gene family which has intriguing implications for the development of Alzheimer's disease in humans.

Age Factors↗