Pulsatile stretch as a novel modulator of amyloid precursor protein processing and associated inflammatory markers in human cerebral endothelial cells.
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ABSTRACT:
SUBMITTER: Gangoda SVS
PROVIDER: S-EPMC5786097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:the exact etiology of sporadic Alzheimer disease (AD) is unclear, but it is interesting that several cardiovascular risk factors are associated with higher incidence of AD. The link between these risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of nitric oxide (NO).to determine the relationship between endothelial derived NO and the expression and processing of amyloid precursor protein (APP).we used human brain microvascular endothelial cells to examine the role of NO in modulating APP expression and processing in vitro. Inhibition of endothelial nitric oxide synthase (eNOS) with the specific NOS inhibitor L-NAME (N(G)-nitro-l-arginine methyl ester) led to increased APP and BACE1 (?-site APP-cleaving enzyme1) protein levels, as well as increased secretion of the amyloidogenic peptide amyloid ? (A?) (control 10.93 ± 0.70 pg/mL; L-NAME 168.21 ± 27.38 pg/mL; P<0.001). To examine the role of NO in modulation of APP expression and processing in vivo, we used brain and cerebral microvessels from eNOS-deficient (eNOS(-/-)) mice. Brain tissue from eNOS(-/-) mice had statistically higher APP and BACE1 protein levels, as well as increased BACE1 enzyme activity and A? (A?(1)(-)(42) wild-type control, 0.737 pg/mg; eNOS(-/-), 1.475 pg/mg; P<0.05), compared with wild-type controls (n=6 to 8 animals per background). Brain microvessels from eNOS(-/-) mice also showed statistically higher BACE1 protein levels as compared with wild-type control.our data suggest that endothelial NO plays an important role in modulating APP expression and processing within the brain and cerebrovasculature. The NO/cGMP pathway may be an important therapeutic target in preventing and treating mild cognitive impairment, as well as AD.
| S-EPMC3064266 | biostudies-literature
Project description:The present study was designed to determine the effects of senescence and angiotensin II (Ang II) on expression and processing of amyloid precursor protein (APP) in human brain microvascular endothelial cells (BMECs). Senescence caused a decrease in APP expression thereby resulting in reduced secretion of soluble APP? (sAPP?). In contrast, ?-site APP cleaving enzyme (BACE1) expression and production of amyloid ? (A?)40 were increased in senescent endothelium. Importantly, in senescent human BMECs, treatment with BACE1 inhibitor IV inhibited A? generation and increased sAPP? production by enhancing a disintegrin and metalloprotease (ADAM)10 expression. Furthermore, Ang II impaired expression of ADAM10 and significantly reduced generation of sAPP? in senescent human BMECs. This inhibitory effect of Ang II was prevented by treatment with BACE1 inhibitor IV. Our results suggest that impairment of ?-processing and shift to amyloidogenic pathway of APP contribute to endothelial dysfunction induced by senescence. Loss of sAPP? in senescent cells treated with Ang II exacerbates detrimental effects of senescence on APP processing. Notably, inhibition of BACE1 has beneficial effects on senescence induced endothelial dysfunction. Reported findings may help to explain contributions of senescent cerebral microvascular endothelium to development of cerebral amyloid angiopathy and Alzheimer's disease (AD) pathology.
| S-EPMC5811245 | biostudies-literature
Project description:The exact physiological function of amyloid-? precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP-/-) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP-/-) and eAPP-/- mice ( P?<?0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP-/- mice and eAPP-/- mice as compared to respective wild-type littermates ( P?<?0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice ( P?<?0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium ( P?<?0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS .
| S-EPMC6168907 | biostudies-literature
Project description:We tested hypothesis that activation of the prostacyclin (PGI2) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI2, iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APPα (sAPPα). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor δ (PPARδ) in human brain microvascular endothelial cells. PPARδ-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPARδ) upregulated ADAM10 and increased production of sAPPα. Genetic deletion of endothelial PPARδ (ePPARδ-/-) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPPα. In vivo treatment with GW501516 increased sAPPα content in hippocampus of wild type mice but not in hippocampus of ePPARδ-/- mice. Our findings identified previously unrecognized role of IP-PPARδ signal transduction pathway in the production of sAPPα in cerebral microvasculature.
| S-EPMC5363732 | biostudies-literature
Project description:Alzheimer�s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP.
| S-EPMC3805747 | biostudies-literature
Project description:Proteolytic processing of the amyloid precursor protein (APP) by the ?- and ?-secretases releases the amyloid-? peptide (A?), which deposits in senile plaques and contributes to the etiology of Alzheimer's disease (AD). The ?-secretase cleaves APP in the A? peptide sequence to generate soluble APP? (sAPP?). Upregulation of ?-secretase activity through the 5-hydroxytryptamine 4 (5-HT4) receptor has been shown to reduce A? production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT4 receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT4 receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT4d receptor-stimulated ?-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for ?-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for ?-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT4 receptor-induced ?-secretase activation.
| S-EPMC3897773 | biostudies-literature
Project description:BackgroundA hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration.New methodWe characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics.ResultsIn BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line.Comparison with existing method(s)In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations.ConclusionsOur evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.
| S-EPMC3931259 | biostudies-literature
Project description:Alzheimer's disease (AD) is the most common cause of dementia among older people, and the prevalence of this disease is estimated to rise quickly in the upcoming years. Unfortunately, almost all of the drug candidates tested for AD until now have failed to exhibit any efficacy. Henceforth, there is an increased necessity to avert and/or slow down the advancement of AD. It is known that one of the major pathological characteristics of AD is the presence of senile plaques (SPs) in the brain. These SPs are composed of aggregated amyloid beta (A?), derived from the amyloid precursor protein (APP). Pharmaceutical companies have conducted a number of studies in order to identify safe and effective anti-A? drugs to combat AD. It is known that ?-, ?-, and ?-secretases are the three proteases that are involved in APP processing. Furthermore, there is a growing interest in these proteases, as they have a contribution to the modulation and production of A?. It has been observed that small compounds can be used to target these important proteases. Indeed, these compounds must satisfy the common strict requirements of a drug candidate targeted for brain penetration and selectivity toward different proteases. In this article, we have focused on the auspicious molecules which are under development for targeting APP-processing enzymes. We have also presented several anti-AD molecules targeting A? accumulation and phosphorylation signaling in APP processing. This review highlights the structure-activity relationship and other physicochemical features of several pharmacological candidates in order to successfully develop new anti-AD drugs.
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Project description:Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25-35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-? region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of ?-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.
| S-EPMC3585261 | biostudies-literature
Project description:Amyloid precursor protein (APP) family members and their proteolytic products are implicated in normal nervous system function and Alzheimer's disease pathogenesis. APP processing and A? secretion are regulated by neuronal activity. Various data suggest that NMDA receptor (NMDAR) activity plays a role in both non-amyloidogenic and amyloidogenic APP processing depending on whether synaptic or extrasynaptic NMDARs are activated, respectively. The APP-interacting FE65 proteins modulate APP trafficking and processing in cell lines, but little is known about their contribution to APP trafficking and processing in neurons, either in vivo or in vitro. In this study, we examined the contribution of the FE65 protein family to APP trafficking and processing in WT and FE65/FE65L1 double knockout neurons under basal conditions and following NMDAR activation. We report that FE65 proteins facilitate neuronal A? secretion without affecting APP fast axonal transport to pre-synaptic terminals. In addition, FE65 proteins facilitate an NMDAR-dependent non-amyloidogenic APP processing pathway. Generation of high-molecular weight (HMW) species bearing an APP C-terminal epitope was also observed following NMDAR activation. These HMW species require proteasomal and calpain activities for their accumulation. Recovery of APP polypeptide fragments from electroeluted HMW species having molecular weights consistent with calpain I cleavage of APP suggests that HMW species are complexes formed from APP metabolic products. Our results indicate that the FE65 proteins contribute to physiological APP processing and accumulation of APP metabolic products resulting from NMDAR activation.
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