Circadian Protein BMAL1 Regulates Astrocyte Activation and Protein Degradation in Health and Disease

The circadian clock regulates gene transcription to control daily rhythms in cellular function, behavior, and disease. Circadian dysfunction is a symptom of aging and neurodegenerative diseases, and recent studies suggest reciprocal regulation between impaired clock function and neurodegeneration. In addition to the clock regulating neuronal activity, non-neuronal glia cells of the brain also possess functional circadian clocks which control their responses to daily oscillations in brain activity, cellular stress, and metabolism. Astrocytes directly support brain function through synaptic interactions, neuronal metabolic support, neuroinflammatory regulation, and control of neurovascular coupling at blood and cerebrospinal fluid (CSF) barriers. Emerging evidence suggests that the astrocyte circadian clock may be involved in many of these processes, and that clock disruption could influence neurodegeneration by disrupting several aspects of astrocyte function.The circadian clock is coordinated by positive and negative mediators that generate transcriptional-translational feedback loops and rhythmic gene expression across time-of-day. At the center of these feedback loops is BMAL1, the only circadian clock gene for which deletion abrogates cellular and behavioral rhythms. Depletion of Bmal1 in astrocytes leads to their cell-type specific activation. Activated glia influence the progression of chronic neurodegenerative conditions such as Alzheimer\u27s disease (AD) through functions that alter the course of pathogenesis and neuroinflammation. Activated glia may be particularly important for limiting or exacerbating the accumulation of toxic protein aggregates common to many neurodegenerative diseases, but studies of the impact of astrocyte activation on protein aggregates have produced conflicting results. Thus, we first investigated how the circadian clock functions in astrocytes to regulate amyloid beta (Aβ) deposition and degradation in models of AD. To address these questions, we generated mice with astrocyte-specific knockout of the master circadian clock gene Bmal1 (BMAL1 aKO mice), which renders astrocytes transcriptionally arrhythmic. We observed that astrocytes not only enter an activated state exhibited by transcriptional markers, but they also differentially express disease-modifying genes. We crossed BMAL1 aKO mice to the APP/PS1 and the APP-NL-G-F/wt models of Aβ accumulation. Similar to the wildtype BMAL1 aKO mice, astrocyte-specific Bmal1 deficiency in both the APP/PS1 and APP-NL-G-F/wt models also strongly increases activation of astrocytes around Aβ plaques but does not affect plaque accumulation or neuronal dystrophy. These results show that while BMAL1 regulates astrocyte activation and transcription, this does not alter Aβ plaque accumulation. More generally, our results demonstrate that the effect of astrocyte activation on Aβ plaque accumulation is likely dependent on the specific transcriptional activation state of the astrocyte. We then investigated how deletion of Bmal1 influences endolysosome function, autophagy, and degradation dynamics of extracellular proteins. In vitro, Bmal1-deficient astrocytes exhibit more endocytosis and degradation of extracellular proteins, lysosome-dependent protein cleavage, and accumulation of LAMP1, RAB7, and acidified autophagosomes. In vivo, BMAL1 aKO brains show accumulation of autophagosome-like structures by electron microscopy. RNA sequencing of isolated astrocytes from young and aged BMAL1 aKO mice indicates broad dysregulation of endolysosome pathways involved in receptor-mediated endocytosis and lysosome function. Since a clear link has been established between neurodegeneration and endolysosome dysfunction over the course of aging, this work provides insight into how the circadian clock may be a master regulator of these crucial astrocyte functions in health and disease. Understanding how the clock regulates astrocyte autophagy and endolysosome function may give insights into methods by which to control not only protein aggregation in neurodegenerative conditions, but likely other disease processes as well

Circadian clock protein BMAL1 broadly influences autophagy and endolysosomal function in astrocytes

An emerging role for the circadian clock in autophagy and lysosome function has opened new avenues for exploration in the field of neurodegeneration. The daily rhythms of circadian clock proteins may coordinate gene expression programs involved not only in daily rhythms but in many cellular processes. In the brain, astrocytes are critical for sensing and responding to extracellular cues to support neurons. The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function but also leads to a unique cell-autonomous activation phenotype. We report here that astrocyte-specific deletion o

Astrocytes deficient in circadian clock gene Bmal1 show enhanced activation responses to amyloid-beta pathology without changing plaque burden

An emerging link between circadian clock function and neurodegeneration has indicated a critical role for the molecular clock in brain health. We previously reported that deletion of the core circadian clock gene Bmal1 abrogates clock function and induces cell-autonomous astrocyte activation. Regulation of astrocyte activation has important implications for protein aggregation, inflammation, and neuronal survival in neurodegenerative conditions such as Alzheimer\u27s disease (AD). Here, we investigated how astrocyte activation induced by Bmal1 deletion regulates astrocyte gene expression, amyloid-beta (Aβ) plaque-associated activation, and plaque deposition. To address these questions, we crossed astrocyte-specific Bmal1 knockout mice (Aldh1l1-Cr

Dural lymphatics regulate clearance of extracellular tau from the CNS

BackgroundAlzheimer's disease is characterized by two main neuropathological hallmarks: extracellular plaques of amyloid- (A) protein and intracellular aggregates of tau protein. Although tau is normally a soluble monomer that bind microtubules, in disease it forms insoluble, hyperphosphorylated aggregates in the cell body. Aside from its role in AD, tau is also involved in several other neurodegenerative disorders collectively called tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), some forms of frontotemporal dementia, and argyrophilic grain disease (AGD). The prion hypothesis suggests that after an initial trigger event, misfolded forms of tau are released into the extracellular space, where they spread through different brain regions, enter cells, and seeding previously normal forms. Thus understanding mechanisms regulating the clearance of extracellular tau from the CNS is important. The discovery of a true lymphatic system in the dura and its potential role in mediating A pathology prompted us to investigate its role in regulating extracellular tau clearance.MethodsTo study clearance of extracellular tau from the brain, we conjugated monomeric human tau with a near-infrared dye cypate, and injected this labeled tau in the parenchyma of both wild-type and K14-VEGFR3-Ig transgenic mice, which lack a functional CNS lymphatic system. Following injection we performed longitudinal imaging using fluorescence molecular tomography (FMT) and quantified fluorescence to calculate clearance of tau from the brain. To complement this, we also measured tau clearance to the periphery by measuring plasma tau in both groups of mice.ResultsOur results show that a significantly higher amount of tau is retained in the brains of K14-VEGFR3-Ig vs. wild type mice at 48 and 72h post-injection and its subsequent clearance to the periphery is delayed. We found that clearance of reference tracer human serum albumin (HSA) was also significantly delayed in the K14-VEGFR3-Ig mice.ConclusionsThe dural lymphatic system appears to play an important role in clearance of extracellular tau, since tau clearance is impaired in the absence of functional lymphatics. Based on our baseline characterization of extracellular tau clearance, future studies are warranted to look at the interaction between tau pathology and efficiency of lymphatic function.Peer reviewe

Returning home: heritage work among the Stl'atl'imx of the Lower Lillooet River Valley

This article focusses on heritage practices in the tensioned landscape of the Stl’atl’imx (pronounced Stat-lee-um) people of the Lower Lillooet River Valley, British Columbia, Canada. Displaced from their traditional territories and cultural traditions through the colonial encounter, they are enacting, challenging and remaking their heritage as part of their long term goal to reclaim their land and return ‘home’. I draw on three examples of their heritage work: graveyard cleaning, the shifting ‘official’/‘unofficial’ heritage of a wagon road, and marshalling of the mountain named Nsvq’ts (pronounced In-SHUCK-ch) in order to illustrate how the past is strategically mobilised in order to substantiate positions in the present. While this paper focusses on heritage in an Indigenous and postcolonial context, I contend that the dynamics of heritage practices outlined here are applicable to all heritage practices

Visual impairment in an optineurin mouse model of primary open-angle glaucoma

Primary open angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. While both 18-month old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG, and may be useful for mechanistic dissection of POAG and therapeutic development

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570