Curcumin Treatment is Associated with Increased Expression of the N-Methyl-D-Aspartate Receptor (NMDAR) Subunit, NR2A, in a Rat PC12 Cell Line Model of Alzheimer’s Disease Treated with the Acetyl Amyloid-β Peptide, Aβ(25–35)

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

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Blood-brain barrier permeability and neuroprotective effects of three main alkaloids from the fruits of Euodia rutaecarpa with MDCK-pHaMDR cell monolayer and PC12 cell line

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2017.12.017 ◽

2018 ◽

Vol 98 ◽

pp. 82-87 ◽

Cited By ~ 10

Author(s):

Yi-Nan Zhang ◽

Yan-Fang Yang ◽

Xiu-Wei Yang

Keyword(s):

Cell Line ◽

Blood Brain Barrier ◽

Pc12 Cell ◽

Cell Monolayer ◽

Brain Barrier ◽

Neuroprotective Effects ◽

Barrier Permeability ◽

Pc12 Cell Line ◽

Blood Brain Barrier Permeability ◽

Brain Barrier Permeability

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Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x211039592 ◽

2021 ◽

pp. 0271678X2110395

Author(s):

Emily C Peters ◽

Michael T Gee ◽

Lukas N Pawlowski ◽

Allison M Kath ◽

Felipe D Polk ◽

...

Keyword(s):

Endothelial Cells ◽

Cerebral Arteries ◽

Amyloid Β ◽

Amyloid Angiopathy ◽

Wild Type ◽

Pial Arteries ◽

Aspartate Receptor ◽

Model Of Alzheimer’S Disease ◽

Intracellular Ca ◽

Cerebral Amyloid

Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid- β(1-40) accumulates around blood vessels. In neurons, amyloid- β impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid- β(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR ( NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid- β(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid- β(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid- β(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid- β(1-40) and impaired in 5x-FAD mice.

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