Summary
Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults, and plays a critical role in brain health and disease. We report that the core clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism, and via a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation in vitro and in vivo, mediated in part by suppression of glutathione-s-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a novel mechanism by which the circadian clock could influence many aspects of brain function and neurologic disease.
Highlights
Circadian disruption promotes astrocyte activation.
Astrocyte-specific deletion of the circadian clock gene BMAL1 induces astrocyte activation.
BMAL1 regulates astrocyte activation by altering glutathione-s-transferase signaling.
Loss of astrocyte BMAL1 enhances neuronal cell death in a co-culture system.
eTOC blurb Lananna et al. show that the circadian clock protein BMAL1 regulates astrocyte activation via a cell autonomous-mechanism involving diminished glutathione-s-transferase signaling. This finding elucidates a novel function of the core circadian clock in astrocytes, and reveals a BMAL1 as a modulator of astrogliosis.
Introduction
Changes in behavioral circadian rhythms are common in a wide range of neurodegenerative diseases (Breen et al., 2014; Hatfield et al., 2004; Morton et al., 2005; Musiek and Holtzman, 2016). Studies suggest that such changes can occur early in disease progression (Breen et al., 2014; Hatfield et al., 2004), sometimes before the onset of any other overt symptoms (Musiek et al., 2018; Tranah et al., 2011). Circadian rhythms are generated by the suprachiasmatic nucleus of the hypothalamus (SCN) (Mohawk et al., 2012), which synchronizes cellular clocks throughout the body to the light-dark cycle. The core molecular clock consists of a transcriptional-translational feedback loop with a positive transcriptional limb, consisting of heterodimers of the bHLH transcription factor BMAL1 (aka ARNTL, aryl hydrocarbon receptor nuclear translocator-like protein 1) with either CLOCK (circadian locomotor output cycles kaput) or NPAS2 (neuronal PAS domain protein 2). The negative limb includes Period (PER), Cryptochrome (CRY), and REV-ERB genes which are transcriptional targets of BMAL1 and provide negative feedback inhibition of BMAL1 function (Mohawk et al., 2012). The core clock regulates transcription of thousands of genes in a highly tissue-specific manner, regulating cellular functions including metabolism, inflammation, and redox homeostasis (Bass and Takahashi, 2010; Zhang et al., 2014a). In addition to disrupted activity and sleep rhythms, dysregulated circadian gene expression patterns are also observed in neurodegenerative disease models (Song et al., 2015; Stevanovic et al., 2017) and patients (Breen et al., 2014; Cronin et al., 2017). However, the impact of clock dysfunction on neurologic diseases of the brain remains poorly understood.
Astrocytes play a critical role in brain health and neurodegenerative disease (Pekny et al., 2016). Dysfunctional astrocytes can drive neurodegeneration (Lian et al., 2015; Macauley et al., 2011; Yamanaka et al., 2008), while optimization of certain astrocyte functions can protect against neurotoxic stimuli (Kraft et al., 2004; Xiao et al., 2014). Astrocyte activation is a ubiquitous response to brain injury, from neurodegeneration to trauma, which has historically been characterized by increased expression of the cytoskeletal protein glial fibrillary acidic protein (GFAP) (Sofroniew, 2014). Recent work has begun to elucidate the diversity of astrocyte activation phenotypes beyond GFAP, as astrocytes activated by different stimuli express unique transcriptional profiles which can be associated with divergent phenotypes, ranging from neurotoxic to neurotrophic (Liddelow et al., 2017; Zamanian et al., 2012). However, a further understanding of astrocyte activation mechanisms is needed in order to effectively target protective responses in astrocytes therapeutically, while minimizing detrimental processes.
Astrocyte activation and circadian clock dysfunction are two pervasive, often co-existent features of neurological diseases, though their interaction is unknown. We previously observed that deletion of the core clock gene Bmal1, which abrogates all circadian clock function, caused severe, spontaneous astrogliosis throughout the mouse brain, which was accompanied by increased oxidative stress, synaptic damage, and inflammation (Musiek et al., 2013). However, the cellular and molecular mechanisms linking BMAL1 to astrocyte activation and function remain uncertain. Thus, we sought to characterize the astrocyte activation induced by circadian clock disruption and evaluate whether the clock protein BMAL1 might regulate astrogliosis in a cell-autonomous manner.
Results
We first asked if non-genetic disruption of circadian rhythms could influence Gfap levels in the brain. Exposure of wild type (WT) C57Bl/6 mice to a 10hr:10hr light:dark cycle induces circadian desynchrony and eventually arrhythmicity, as well as loss of detectable circadian transcript oscillations in the cerebral cortex (Fig. 1A and S1A). After 6 weeks of circadian disruption, Gfap transcript levels were increased throughout the circadian cycle (Fig. S1B), with an average increase of 19.7% (Fig. 1B). Thus, light-induced behavioral circadian rhythm disruption promotes astrocyte activation.
Circadian clock function can be abrogated by deletion of the critical clock gene Bmal1. We previously observed that constitutive or post-natal global deletion of Bmal1 in mice induced striking increases in GFAP+ astrocytes throughout the brain (Musiek et al., 2013; Yang et al., 2016). We asked if the astrocyte activation observed following Bmal1 deletion was simply a response to neuronal injury, or a cell-autonomous process regulated by the core clock in astrocytes. To address this, we examined the brains of neuron-specific Bmal1 KO mice (Camk2a-iCre;Bmal1f/f). These mice express Cre recombinase in a pan-neuronal manner under the control of a BAC-CaMKIIa promoter, yielding widespread neuronal deletion of Bmal1 (Fig. S1C) and loss of behavioral circadian rhythms (Izumo et al., 2014). At 4mo, Camk2aiCre+;Bmal1f/f mice exhibited a modest increase in astrocyte activation, as assessed by GFAP immunostaining (Fig. 1C,D) and qPCR (Fig. 1E), in the cortex and hippocampus. However, similarly-aged Nestin-Cre+;Bmal1f/f (referred to as NBmal1 KO) mice, in which Bmal1 is deleted in neurons and astrocytes, have much more prominent astrogliosis throughout the brain and higher expression of activation-related transcripts Aqp4 and Serpina3n (Fig. 1C-E). While NBmal1 KO mice exhibit large increases in GFAP+ astrocytes in the cortex, this did not appear to be due to increased astrocyte division, as NBmal1 KO mice had no significant increase in dividing Ki67+, GFAP+ double-positive cells (Fig. S1D), or in immunoreactivity for the panastrocyte marker S100B (Fig. S1E). This finding suggests that while a small component of astrogliosis observed following Bmal1 deletion may be a response to neuronal injury, BMAL1 within astrocytes appears to play a critical cell-autonomous role in astrocyte activation.
To assess the potential cell-autonomous effects of Bmal1 on astrocyte activation, we generated primary astrocyte-enriched cultures from Bmal1 KO mice and WT littermates, and examined activation markers in vitro. Bmal1 KO astrocytes were viable and appeared to have normal morphology, but had significantly elevated expression of Gfap and Aqp4 mRNA and GFAP protein (Fig. 2A,B), suggesting spontaneous activation in the absence of neuronal influence. In order to exclude a developmental influence of BMAL1, we used siRNA to knock down Bmal1 expression in WT primary astrocyte cultures. This method resulted in a 95% decrease in BMAL1 protein (Fig. 2C,D) and a 66% decrease in expression of the BMAL1 target Nr1d1 (which encodes REV-ERBα)(Fig. S2A). Bmal1 knockdown (KD) in astrocytes induced a highly consistent (across 14 independent experiments) 36% increase in Gfap mRNA expression (Fig. S2A) as well as a 68% increase in GFAP protein (Fig. 2C,D). Bmal1 KD in primary astrocytes from Per2-luciferase reporter mice caused a loss of circadian Per2 rhythms (Fig. 2E). We also saw a significant increases in the astrocyte activation marker S100a6 (1.7 fold), the cytokine Il6 (2.4 fold), and Il33 (1.6 fold) (Fig. S2A), an astrocytic cytokine implicated in astrocyte-microglia signaling (Wicher et al., 2017). Thus, disruption of circadian function via knockdown of Bmal1 in WT astrocytes induces a cell-autonomous reactive phenotype in vitro.
We next sought to confirm these findings by specifically deleting Bmal1 in astrocytes in vivo. We found that intracerebroventricular injection of an AAV8 viral vector expressing eGFP under a Gfap promoter (AAV8-GFAP-eGFP) on postnatal day 2 led to widespread eGFP expression in resting astrocytes throughout the cerebral cortex, with almost no neuronal labeling (Fig. 2F,G, S2B). Thus, we injected P2 Bmal1f/f mice either with AAV8-GFAP-GFP, or with an identical virus expressing a Cre-GFP fusion protein (AAV8-GFAP-CreGFP). At 5mo, we observed Bmal1 deletion and increased GFAP expression specifically in astrocytes expressing AAV8-GFAP-CreGFP (Fig. 2F). It should be noted that the fusion product of the GFAP-CreGFP vector exhibits very weak nuclei-restricted fluorescence, making it much more difficult to see than that of the AAV8-GFAP-eGFP vector. Mice treated with the eGFP control vector had widespread astrocyte labeling throughout the cingulate and retrosplenial cortices, intact BMAL1 expression, and very little astrocyte activation as defined by GFAP immunoreactivity (Fig. 2F,G). Mice injected with the CreGFP vector showed astrocytic nuclear GFP expression and 3.4- and 3.1-fold increases in GFAP immunoreactivity in the cingulate and retrosplenial cortices, respectively (Fig. 2G). Finally, we generated tamoxifen-inducible astrocyte-specific Bmal1 KO mice using an Aldh1l1-CreERT2 driver line (Aldh1l1-CreERT2;Bmal1f/f mice) (Srinivasan et al., 2016). We treated Cre+ and Cre- mice with tamoxifen at 1mo, and examined astrocyte activation at 3mo. We observed specific deletion of Bmal1 in astrocyte nuclei of the hippocampus and cortex (Fig. S2D,E). Aldh1l1-Cre+ mice had significant increases in astrogliosis, as assessed by GFAP immunostaining in the hippocampus (Fig. 2H) and a nearly significant (p=0.055) increase in the retrosplenial cortex (Fig. S2C). While it is difficult to directly compare tamoxifen-inducible and constitutive Cre lines, Aldh1l1-Cre+ mice had similar levels of astrogliosis to tamoxifen inducible global Bmal1 KO mice (CAG-CreERT2+;Bmal1f/f)(Yang et al., 2016) also harvested 2mo after TAM treatment (Fig. 2H, S2C). Astrogliosis was not caused by Cre expression, as Cre+;Bmal1flox/wt mice had no phenotype (Fig. 2H, S2C). Ald1l1-Cre+ mice also had spontaneous and significant increases in a number of additional astrocyte activation marker transcripts including S100a6 (2.5 fold), C4b (2.6 fold), Cxcl5 (7.5 fold), and Mmp14 (3.1 fold) (Fig. S2F). In total, these experiments demonstrate that Bmal1 deletion in astrocytes can induce activation in a cell-autonomous manner.
While activated astrocytes generally exhibit morphologic changes and increased GFAP expression, recent studies have demonstrated that the transcriptional profile of activated astrocytes is heterogeneous and can define their function (Liddelow et al., 2017; Zamanian et al., 2012). In order to examine the transcriptional profile in the brain following Bmal1 deletion, we performed microarray analysis of cortical tissue from 11mo NBmal1 KO mice, Cre-controls, and Per1Brdm/Per2Brdm (Per1/2mut) mice, placed in constant dark conditions for 36 hours then harvested at CT 6:00 and 18:00. Per1/2mut mice were included because they are behaviorally arrhythmic and have a dysfunctional circadian clock, but have no astrogliosis phenotype as well as tonically increased levels of BMAL1 transcriptional targets (Fig. 3A, S3A). To understand astrocyte transcriptional changes in NBmal1 KO brain, we examined the overlap between the 200 most upregulated transcripts in NBmal1 KO, with the 200 most astrocyte-specific transcripts, according to the Barres Lab RNAseq database (Zhang et al., 2014b) (Fig. 3A). Thirty genes overlapped, including Gfap, Aqp4 (encoding the astrocytic water channel Aquaporin-4), and Megf10, a gene involved in astrocyte-mediated synaptic elimination (Chung et al., 2013). C4b, a complement cascade component also implicated in synapse elimination and a marker of astrocyte activation and aging (Boisvert et al., 2018; Clarke et al., 2018), was strongly upregulated (4-fold) in NBmal1 KO brain. Several other disease-associated astrocytic genes were increased in NBmal1 KO brain, including inflammatory mediators such as Cxcl5 (Bortell et al., 2017) and Ccr7 (Gomez-Nicola et al., 2010), calcium binding protein genes S100a4 (Dmytriyeva et al., 2012) and S100a6 (Hoyaux et al., 2002), and matrix metaloproteinases Mmp14 (Rathke-Hartlieb et al., 2000) and Mmp2 (Li et al., 2011). Nearly all of these transcripts were either unchanged or decreased in Per1/2mut mice, demonstrating the key role of decreased BMAL1 function in this phenotype. We next performed microfluidic qPCR profiling of global Bmal1 KO hippocampal samples for markers of astrocyte activation and polarization (Liddelow et al., 2017), which demonstrated that most pan-reactive markers were increased, while Bmal1 deletion did not clearly polarize astrocytes toward an neurotoxic or neurotrophic profile (Fig. 3B). Taken together, these genetic profiling studies suggest that Bmal1 deletion induces a unique state of astrocyte activation with potential implications for astrocyte behavior in disease states.
Although Per1/2mut and NBmal1 KO mice are both behaviorally arrhythmic, expression of BMAL1 transcriptional targets Nr1d1 and Dbp are elevated solely in the Per1/2mut mice, due to loss of inhibition of BMAL1 (Fig. 3A, S3A). Gfap is not elevated Per1/2mut mice (Fig. 3A, S3A), suggesting that loss of BMAL1 activity, or at least blunting of BMAL1 oscillation (as in Fig. 1A), is needed to induce astrocyte activation. In order to determine if Gfap and other selected activation markers are, in fact, clock controlled genes, we harvested cortex from 4mo NBmal1 KO and Cre- controls at 4-hour intervals across one circadian cycle in constant darkness. Gfap mRNA levels showed no circadian oscillation in control or Cre+ mice, and were elevated across the circadian cycle in the cortex of Cre+ mice. These findings were duplicated in other markers of astrocyte activation, including Aqp4 and Mmp14 (Fig. S3B). The lack of circadian oscillation suggests that this astrogliosis phenotype is not due to direct regulation of Gfap and other activation markers by the clock.
To address potential mechanisms linking BMAL1 to astrocyte activation, we performed pathway analysis on our NBmal1 KO transcriptomic data using two bioinformatics tools: Ingenuity Pathway Analysis (IPA) and DAVID Bioinformatics Resources 6.8. We analyzed an inclusive gene list consisting of all genes that differed between control and NBmal1 KO at 6pm with uncorrected p values<0.01 (Table S1). IPA Canonical Pathway Analysis identified Glutathione-mediated detoxification as the top hit (Fig. 3C, p= 0.000013). Functional Annotation Clustering with DAVID showed that the most enriched gene cluster focused on glutathione-s-transferase (GST) activity, and 4 of the top 5 KEGG pathways identified were related to glutathione metabolism (Fig. S3C). GST enzymes catalyze the phase II detoxification of reactive intermediates in cells via adduction to glutathione and can alter cellular signaling via glutathionylation of proteins (Grek et al., 2013). We found that expression of numerous GST isoforms was suppressed in NBmal1 KO cortex, and were increased in Per1/2mut cortex (Fig. 3D), suggesting reciprocal regulation by the positive and negative limbs of the circadian clock. In support of our microarray analysis, we observed significant decreases in 2 of the 3 GST isoforms probed in cortex from 3mo CAG-Cre+;Bmal1f/f mice 9 days after tamoxifen treatment (Fig. S3D) and in 1 of the 3 GST isoforms probed in hippocampus from 3mo Aldh1l1-Cre+ mice 2 months after tamoxifen treatment (Fig. S3E). We next asked if altered glutathione homeostasis within astrocytes linked Bmal1 deficiency with astrocyte activation. Treatment with N-acetyl cysteine (NAC), a glutathione precursor, completely prevented increased Gfap expression in astrocytes following Bmal1 knockdown (Fig. 3E). Conversely, incubation of WT astrocytes with buthionine sulfoxime (BSO), which blocks cysteine uptake and depletes cellular glutathione levels, lowered cellular glutathione levels (Fig. S3F) and caused a similar increase in Gfap mRNA to that observed with Bmal1 knockdown (Fig. 3E). Treatment of Bmal1 deficient astrocytes with BSO did not induce a further increase in Gfap suggesting that BSO and Bmal1 knockdown are causing increases in Gfap through overlapping pathways. Interestingly, neither Bmal1 KO nor knockdown of Bmal1 in cultured primary astrocytes altered glutathione levels (Fig. S3F). Additionally, the oxidation state of glutathione (ratio of reduced GSH to oxidized GSSG) was not altered in cortex from NBmal1 KO or Per1/2mut mice (Fig. S3G). As glutathione levels and oxidation state remained unchanged with loss of Bmal1, we next examined glutathione signaling via glutathionylation, which has been shown to oscillate in a circadian manner in the SCN (Wang et al., 2012). Western blot analysis of siSCR and siBmal1 transfected WT astrocytes treated with biotin-linked glutathione ethyl ester (bioGEE) revealed widespread changes in the pattern of s-glutathionylated proteins in Bmal1 KD cells, which are similar to changes seen in BSO treated controls (Fig. 3F). In general, numerous bands showed increased biotin labeling, indicating decreased glutathionylation, as would be expected with decreased GST expression. These data suggest that the observed astrogliosis may be due at least in part to altered protein glutathionylation resulting from impaired expression of specific GSTs.
We next examined whether bolstering glutathione signaling can prevent astrogliosis following Bmal1 deletion in vivo. We previously showed that tamoxifen-inducible global Bmal1 deletion in mice (CAG-Cre+;Bmal1f/f mice) causes astrocyte activation (Yang et al., 2016). We treated CAG-Cre+;Bmal1f/f mice with NAC in their drinking water (40mM beginning 5 days before tamoxifen) and via intraperitoneal injection (150mg/kg, daily beginning 2 days before tamoxifen), and induced Bmal1 deletion via tamoxifen administration for 5 days. Both administrations of NAC were continued for the duration of the experiment. Cortex was harvested 9 days after the start of tamoxifen treatment and analyzed by qPCR. Tamoxifen treatment induced an 87% loss in Bmal1 and a 79% loss in expression of the Bmal1 target Nr1d1 (Fig. S3H), as well as a 64% increase if Gfap expression (Fig. 3G). Administration of NAC was able to provide a significant, but partial rescue of the Gfap increase seen in CAG-Cre+;Bmal1f/f animals (Fig. 3G). These results suggest that Bmal1 regulates Gfap expression at least in part through modulation of glutathione signaling in vivo.
Finally, we examined potential pathogenic consequences of Bmal1 deletion in astrocytes. We generated primary astrocyte-enriched cultures from CAG-Cre+;Bmal1f/f mice and Crelittermates and treated them with tamoxifen in vitro after culturing to delete Bmal1. These cultures are primarily astrocytes, but this methods yields cultures which contain ~2% microglia and/or oligodendrocyte precursor cells (Schildge et al., 2013). Primary WT cortical neurons were then co-cultured on these astrocytes for up to 12 days and a subset were subjected to oxidative stress by hydrogen peroxide exposure (100µM H2O2) for 24 hours (Fig. 4A). We found no difference in the number of neurons surviving on Cre- and Cre+ astrocyte cultures 1 day after plating (as quantified by staining for neuronal nuclei with NeuN, as these cells do not yet have many MAP2 positive neurites) (Fig. 4B). At day 7 after plating, there was a 45% decrease in the number of NeuN+ nuclei (Fig. 4B,C), a trend toward a decrease in MAP2 staining, and a trend toward an increase in cleaved-caspase-3 staining (Fig. S4A,B) in the Cre+ astrocyte condition. The decrease in MAP2 staining became significant after 12 days (Fig. 4D,E). H2O2 exposure at day 12 decreased MAP2 levels in Cre- cultures, and the combination of Bmal1 deletion and H2O2 killed nearly all neurons (Fig. 4D,E). This finding shows that Bmal1-deficient astrocytes are less able to sustain neuronal viability, particularly in the face of oxidative stress.
Discussion
Despite a large body of evidence describing circadian disruptions in both aging and neurodegenerative disease, little is known about the implications of circadian dysfunction on the brain. Here, we provide evidence that behavioral circadian disruption or disruption of the astrocytic molecular clock via manipulations of the master clock gene Bmal1 induce astrogliosis and astrocyte dysfunction. The critical, but complex role of astrocytes in brain pathologies and neuroinflammation underscores the importance of elucidating both triggers and mechanisms of astrocyte activation, and our data illuminate BMAL1 as a potent regulator of astrocyte activation by both cell-autonomous and non-autonomous mechanisms.
The existence of the astrocyte circadian clock has been well documented (Prolo et al., 2005), and the critical role of the astrocyte clock in maintaining circadian rhythms in the SCN has recently been described (Barca-Mayo et al., 2017; Brancaccio et al., 2017; Tso et al., 2017). Clock genes have been shown to regulate astrocytic glutamate uptake and ATP release (Beaule et al., 2009; Marpegan et al., 2011), but the function of the core clock in astrocytes is otherwise unexplored. We have shown here, using several in vitro and in vivo methods, that loss of Bmal1 function in astrocytes causes cell-autonomous astrocyte activation. However, deletion of Bmal1 specifically in neurons also induces a partial astrocyte activation phenotype, suggesting a coexistent cell non-autonomous mechanism. We have previously shown that Bmal1 deletion in cultured neurons causes toxicity (Musiek et al., 2013), implicating a possible damage signal from Bmal1-deficient neurons which activates nearby astrocytes. Alternatively, the neuron-specific Bmal1 KO mice used in this study are behaviorally arrhythmic (Izumo et al., 2014), and this loss of rhythms can induce astrocyte activation, as seen in 10h:10h L:D exposed animals (Fig. 1). Thus, cell non-autonomous influences on glial activation in the brain must be examined in the future.
One important question is whether the loss of rhythmic function of the circadian clock plays a key role in this phenotype, or if this is a “non-circadian” function of BMAL1. Our data suggest that arrhythmicity in the setting of increased BMAL1 expression, as in Per1/2mut mice, does not induce astrocyte activation, as these mice express increased levels of GSTs. Thus, astrocyte activation appears to be dependent on suppression of BMAL1-mediated transcription. However, BMAL1 DNA binding is highly rhythmic and regulated by the clock (Koike et al., 2012), making it difficult to separate entirely from circadian rhythms. Bmal1 knockdown in astrocytes abrogates circadian Per2-luc oscillations, which could contribute to increased Gfap expression in cell culture. Moreover, we show in Fig. 1 that circadian disruption by exposure of mice to 10h:10h L:D conditions, which blunts Bmal1 oscillations in the brain, can induce Gfap expression. Thus, dampening the rhythmicity of BMAL1 expression through circadian clock disruption likely plays a role in mediating astrogliosis, perhaps by restricting BMAL1 levels at certain key times of day. BMAL1 levels can be suppressed or blunted by several factors related to neurodegeneration, such as aging (Wyse and Coogan, 2010), inflammation (Curtis et al., 2015), or amyloid-beta (Song et al., 2015), all of which could potentially influence astrocyte activation and function in a BMAL1-dependent manner.
The activation profile induced in Bmal1-deficient astrocytes encompasses upregulation of a variety of genes across the pre-defined neurotoxic, neurotrophic, and pan-reactive categories (Liddelow et al., 2017). However, it is important to remember that these astrocyte phenotypes are thought to be induced by cytokine release from microglia (Liddelow et al., 2017), whereas activation due to loss of Bmal1 can be cell-autonomous. Our findings contrast with those of Nakazato et al, who found that S100b-Cre+;Bmal1f/f mice did not develop astrocyte activation, and attributed the astrocyte activation phenotype of Nestin-Cre+;Bmal1f/f mice to pericyte dysfunction (Nakazato et al., 2017). However, the degree of astrocyte Bmal1 deletion in the S100b-Cre mouse was not demonstrated in that study. While our data does not exclude a contribution of blood-brain barrier dysfunction to the Nestin-Bmal1 phenotype, we provide multiple lines of evidence demonstrating cell-autonomous astrocyte activation. While Bmal1-deficient astrocytes do not assume a clear neurotoxic (“A1”) phenotype, our data demonstrate that they express many activation and pro-inflammatory markers, and that they are less able to support neuronal survival in a co-culture system. In addition to Gfap, C4b, a complement component and marker of astrocyte activation and aging (Boisvert et al., 2018; Clarke et al., 2018), is strongly increased following Bmal1 deletion, as are several other pan-reactive markers including Timp1 and Serpina3n (Liddelow et al., 2017). Increased expression of several genes implicated in neuroinflammation and neurodegeneration, including Megf10 (Chung et al., 2013; Iram et al., 2016), which mediates astrocytic synapse elimination, and Pla2g3, a phospholipase implicated in ROS-induced neuronal damage (Martinez-Garcia et al., 2010) was observed. Activation marker Lcn2 (Lipocalin 2), an anti-inflammatory mediator in the brain (Kang et al., 2017), was not increased, again suggesting a pro-inflammatory state.
Our data support a mechanism in which loss of BMAL1 induces astrogliosis through disruption of GST-mediated protein glutathionylation. In addition to its role in quenching oxidative stress, glutathione can form adducts on cysteine residues in a process termed glutathionylation, which is catalyzed by glutathione-s-transferases (Grek et al., 2013). Glutathionylation can serve as a signaling mechanism in processes such as mitochondrial metabolism (Mailloux and Treberg, 2016) and apoptosis (Naoi et al., 2008). Indeed, circadian rhythms of protein s-glutathionylation in the SCN may regulate neuronal excitability (Wang et al., 2012). Because Bmal1-deficient astrocytes have normal total glutathione levels, it is likely that disruption of GST-mediated glutathione signaling or utilization, not glutathione depletion, mediates BMAL1-controlled astrogliosis. Accordingly, we observed an altered pattern of protein s-glutathionylation in Bmal1-deficient astrocytes, which resembled that seen in BSO-treated cells. Thus, supplementing Bmal1–deficient astrocyte cultures or mice with NAC presumably prevents astrogliosis by promoting non-enzymatic glutathionylation of protein targets, thereby circumventing the loss of GSTs (Grek et al., 2013). Further studies are needed to identify specific glutathionylation targets that mediate astrocyte activation, and to understand regulation of astrocyte glutathione signaling in health and disease.
In summary, our data identify the circadian clock protein BMAL1 as a unique, cell-autonomous regulator of astrogliosis and posit altered GST-mediated protein glutathionylation as a mediator of this effect. Our findings also show that non-genetic circadian disruption promotes Gfap expression, and demonstrate that Bmal1-deficient astrocytes have diminished neurotrophic function and impaired engagement of amyloid plaques. BMAL1 serves as a novel link between the core circadian clock and astrogliosis, and this study provides new insights into how the circadian clock might influence neurodegeneration.
Acknowledgements
The authors declare no financial conflicts of interest. This research was funding by NINDS grant K08NS079405 (ESM), NIA grant R01AG054517 (ESM), and a New Investigator Research Grant from the Alzheimer’s Association (ESM). JST is an Investigator in the Howard Hughes Medical Institute. SAL was supported by a postdoctoral fellowship from the Australian National Health and Medical Research Council (GNT1052961), the Glenn Foundation Glenn Award, an anonymous donation, and the generous support of V. and S. Coates.
Footnotes
Lead Contact: Erik Musiek