Basic Science and Pathogenesis.

Background: Cerebral microvascular dysfunction and nitro-oxidative stress are present in patients with Alzheimer's disease (AD) and may contribute to disease progression and severity. A pro-nitro-oxidative environment can lead to post-translational modifications of ion channels central to microvascular regulation in the brain, including the large conductance Ca-activated K channels (BK). Nitro-oxidative modulation of BK can resulting in decreased activity and vascular hyper-contractility, thus compromising neurovascular regulation. We hypothesized that nitro-oxidation of vascular BK function blunts micro- and neuro-vascular responses in 5x-FAD mice.

Methods: Six-months-old female 5x-FAD and wild-type (WT) littermates were used. Brain lysates were used to quantify BK nitro-oxidation. Cerebral arteries lysates were used to assess BK subunit expression by qPCR. A different set of cerebral arteries were used for patch clamp electrophysiology assessment of BK currents, ex vivo pressure myography and quantification of calcium (Ca) sparks in smooth muscle cells (SMC) by spinning-disk confocal microscopy. Basal cerebral perfusion and neurovascular coupling were assessed by laser speckle contrast imaging. Differences between means were tested using t-tests and considered statistically significant when P<0.05.

Results: We observed that BK S-nitrosylation, a proxy for nitro-oxidative modification, was increased in the brain of AD patients and in 5x-FAD females. In functional experiments, we observed that BK currents were lower in cerebral artery SMC of 5x-FAD than in WT. As a consequence, microvascular reactivity to BK blockade (iberiotoxin, 30 nM) was blunted in 5x-FAD, suggesting lower basal BK activity. This reduction in microvascular BK activity was independent of SMC Ca sparks or BK mRNA expression. We then performed rescue experiments by incubating membrane patches and pressurized arteries with the reducing agent dithiothreitol (DTT, 10 µM), and observed that DTT restored BK currents and sensitivity to iberiotoxin in cerebral artery SMC from 5x-FAD. In vivo experiments showed that basal perfusion to the frontal cortex and neurovascular coupling were lower in 5x-FAD when compared to WT littermates.

Conclusion: These data suggest that excessive nitro-oxidation of BK mediates neuro- and micro-vascular impairments in female 5x-FAD mice, and that this process is reversible and can be rescued by reducing agents.

Funding: National Institutes on Aging and Alzheimer's Association.