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Effects of sleep deprivation and adrenergic inhibition on glymphatic flow in humans

Periodic Reporting for period 1 - Human Glymphatics (Effects of sleep deprivation and adrenergic inhibition on glymphatic flow in humans)

Reporting period: 2018-04-01 to 2020-03-31

Sleep problems and disordered sleep is highly prevalent in modern society and may lead to a range of diseases including obesity, cardiovascular problems and Alzheimer’s Disease. Nevertheless, the molecular and biological underpinnings of why we sleep remain poorly understood. Recent preclinical evidence show that while rodents are asleep, cardiovascular pulsations driven an influx of cerebrospinal fluid into the brain parenchyma that generates a connective fluid flow that effectively promotes the removal of metabolic by-products that has accumulated during the day. This sleep dependent macroscopic pathway may be one of the reasons why we need to sleep and why insufficient sleep is detrimental to the organisms.

The aim of this project is to investigate whether such macroscopic clearance exists in the human brain during sleep and whether it has relevant biological consequences for performance and cognition. To investigate these questions, we set up a human sleep study using novel, state of the art ultra-fast magnetic resonance imaging (MRI) protocols, including multiband and magnetic resonance encephalography (MREG). These MR sequences enabled us to acquire images of the entire human brain at 5-10 times each second. By applying these novel imaging methods I aimed at: 1. addressing how cardiac driven brain pulsations in the brain change between sleep and wakefulness; 2. whether levels of brain pulsations are associated with sleep intensity; and 3. Whether the brain pulsations and clearance can be enhanced by adrenergic antagonists similar to what has been shown in rodents.

Secondly, while data collection for the primary study was ongoing, we investigated data related to brain clearance in an already collected cohort of healthy controls who underwent a controlled sleep deprivation protocol. The protein transporter aquaporin-4 has been shown to play a critical role in facilitating the influx of CSF from perivascular spaces during sleep. We therefore set out to investigate whether common genetic variants of the aquaporin-4 gene modulate sleep and sleep-wake behavior in this cohort, which would provide further evidence of a sleep driven and aquaporin-4 dependent pathway in humans.
"The project started with the development of the clinical sleep study protocol and the approval of the design and method by the ethical protocol in Copenhagen. Following approvals, data collection was initiated which included the data collection of 22 healthy human subjects across three brain pulsation imaging sessions, at baseline (8h awake), sleep deprived (32h awake) and after sleep deprivation with an adrenergic challenge (32 h awake + carvedilol 25mg). The protocol was carried out in a double blind, placebo controlled manner and all planed subjects have been successfully recorded. Due to the complex protocol, data collection and pre-processing took longer than anticipated, which has meant that data analysis is currently ongoing. A major obstacle has been the removal of MR induced artifacts on the EEG data collected during imaging. These EEG data are central for the interpretation of the collected data, as they allow us to quantify sleep intensity during imaging. Based on extensive data processing and visual sleep scoring from two blinded sleep clinicians, we recently marked this major milestone as complete [A1].

Simultaneous with data collection in the sleep study, we worked on an already existing data set on 123 healthy controls genotyped for a common variant of aquaporin-4 (AQP4). The data provided novel evidence that subjects carrying the low AQP4-expressing variant had enhanced sleep intensity and where able to cope slightly better with prolonged wakefulness. Given how associated AQP4 variants have also been associated with cognitive decline in Alzheimer’s patients the data may provide a tantalizing link between Alzheimer’s disease, sleep intensity, and AQP4 in humans. Although future studies are warranted, this association may suggest that cerebrospinal flow through AQP4 may be linked to brain clearance in humans [Figure, A2].

Due to MREG and ultra-fast fMRI collaboration with the Kiviniemi lab in Oulu Finland, we are currently working on two additional manuscripts where we detect sleep and Alzheimer’s disease using MREG imaging [A3 & A5].

Finally, as part of my efforts to expand the knowledge of the glymphatic system in humans, we investigated the role of NE antagonists, including beta-blockers, on the risk of developing Alzheimer’s disease in the Danish national registry. Because commonly used beta-blocker drugs vary in their ability to permeate the blood-brain barrier, we compared Alzheimer’s risk across patient consuming high and low blood-brain barrier penetrant beta-blockers. Our novel data show that in a population of hypertensive patients, high blood-brain barrier penetrant beta-blockers convey a 26% reduced risk for AD compared to low blood-brain barrier penetrant beta-blockers, matching the hypothesis of a CSF driven glymphatic pathway that can be enhanced by NE antagonists [A4].

Manuscripts:
[A1, in preparation] Holst et al., “Human brain pulsations during NREM sleep and their modulation by sleep deprivation and adrenergic antagonists”
[A2, Published] Ulv Larsen, …, Holst. ""Haplotype of the astrocytic water channel AQP4 is associated with slow wave energy regulation in human NREM sleep."" PLoS Biology 18.5 (2020): e3000623.; doi.org/10.1371/journal.pbio.3000623
[A3, in submission] Helakari, … Holst, … Kiviniemi. “Sleep-specific changes in physiological brain pulsations”
[A4, in preparation] Beaman, …, Holst. “Effect of blood-brain barrier penetrance on the risk of Alzheimer’s disease amongst β-blocker patients: A nation-wide cohort study”
[A5, in submission] Rajna, … Holst, … Kiviniemi. “Cardiovascular brain impulse in Alzheimer’s disease”"
The research project has made several original contributions in neurobiology and sleep research. Because data analyses are still ongoing, the final impact of the project remains to be determined. Nevertheless, the recent publication in PLOS Biology had substantial impact to the field and was picked up in a number of news outlets and general public media. We expect the final publications currently in preparation to have a similar if not greater impact to the field.
Secondly, as part of the project, I was able to organize and chair a symposium session at the 32nd European College of Neuropsychopharmacology Congress in Copenhagen entitled “The glymphatic system, an uncharted framework in sleep medicine and neurodegenerative disease”. The session was well attended and we received many interesting questions by peers in neuroscience and pharma research.
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