Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-b (Ab) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-b (oAb 42 ), interacting with a7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAb 42 activates both homomeric a7and heteromeric a7b2-nAChR subtypes while preferentially enhancing a7b2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAb 42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/ PS1 transgenic mice, genetically null for the b2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for a7b2-nAChR in mediating the effects of oAb 42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of a7b2-nAChR in oAb 42 -induced cognitive decline.
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.
SLC 4A11 is a multifunctional membrane transporter involved with H + transport, NH 3 and alkaline pH stimulated H + transport, and water transport. The role of SLC 4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC 4A11/LacZ staining is primarily in the long‐looped descending thin limb ( DTL ) as determined by colocalization with aquaporin 1 ( AQP 1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC 4A11 in the upper DTL s (which are AQP 1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTL s (which are AQP ‐1‐null). SLC 4A11 also colocalized with AQP 1 and the urea transporter UT ‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC 4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC 4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTL s. SLC 4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC 4A11 pathways in mouse and rat and complement previous studies of DTL Na + , urea and water permeability indicating that the upper and lower DTL s of long‐looped nephrons are functionally distinct.
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Introduction: Cerebral amyloid angiopathy (CAA), the accumulation of amyloid- β (1-40) (A β ) around cerebral arteries, impairs endothelial function. Endothelium-dependent dilation is a consequence of transient increases in intracellular [Ca 2+ ] in endothelial cells (EC). The Ca 2+ permeable N-methyl-D-aspartate receptor (NMDAR) mediates endothelium-dependent dilation, although if these effects are dependent on Ca 2+ influx and transients, or if they are impaired by A β , remains undetermined. Hypothesis: A β inhibits endothelial NMDAR-mediated Ca 2+ influx and transients in murine pial arteries. Methods: We performed Ca 2+ time-lapse imaging of en face pial arteries from cdh5-GCaMP8 mice to quantify EC Ca 2+ events induced by NMDAR activation. Data are means ± SEM. Results: Elemental Ca 2+ entry through NMDAR, hereon called NMDAR sparklets , was assessed in arteries incubated with EGTA-AM and cyclopiazonic acid (CPA) to inhibit intracellular Ca 2+ transients. NMDA (10 μM) induced an increase in NMDAR sparklets frequency when compared to vehicle, an effect inhibited by the NMDAR antagonist D-APV (in Hz: 0.12±0.01 vs 0.44±0.05 vs 0.21±0.02, vehicle vs NMDA vs NMDA+D-APV, p<0.05). Further, pial arteries exposed to NMDA without EGTA-AM and CPA showed an increase in the frequency of intracellular Ca 2+ transients, also blocked by D-APV (in Hz: 0.24±0.05 vs 0.53±0.10 vs 0.28±0.05, vehicle vs NMDA vs NMDA+D-APV, p<0.05). We then tested the effects of A β on Ca 2+ events in pial artery EC. We observed that 30 minutes exposure to A β (5 μM) caused a significant reduction in NMDAR sparklets (in Hz: 0.62±0.07 vs 0.22±0.03, NMDA vs NMDA + A β , p<0.05) but did not significantly alter intracellular Ca 2+ transients (in Hz: 0.62±0.37 vs 0.27±0.07, NMDA vs NMDA + A β ). Lastly, we performed pressure myography on pial arteries of wild-type and 5x-FAD mice, a model of familial Alzheimer’s disease with rapid amyloid accumulation. 5x-FAD mice displayed impaired vasodilation to NMDA (vasodilation (%): 9.86±0.64 vs 4.22±2.76, wild-type vs 5x-FAD , p<0.05). Conclusion: These preliminary data suggest that A β impairs endothelial NMDAR-associated Ca 2+ influx events in cerebral arteries, which can impair blood flow in CAA patients, thus contributing to cognitive impairment.
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