Background
Glucocorticoids (GCs) are the primary hormones released from the adrenal gland in response to stressful events. Stress increases circulating levels of endogenous GCs (cortisol in humans and corticosterone in rodents) [
1], which in turn may induce neurodegenerative diseases, such as Alzheimer’s disease (AD) and depression vulnerability [
2,
3]. Growing data showed that prolonged stress and chronic GC exposure produced abnormal behaviors in experimental animals and increased risk of psychiatric disorders in humans, for example, chronic stress plays an important role in the etiology of sporadic AD [
4‐
7]. Furthermore, stress-level GCs are known to reduce hippocampal dendritic complexity [
8,
9] and promote hippocampal neurons injury [
10]. These studies suggest that chronic exposure to stress-level GCs results in neuronal injury and contributes to the development of neurodegenerative diseases, but the precise molecular and cellular mechanisms remain to be fully elucidated.
An emerging literature suggests that neuroinflammation plays an important role in many neurological diseases such as Parkinson’s disease (PD) and AD [
11,
12]. GCs have been traditionally appreciated for their potent anti-inflammatory properties, but growing investigation has revealed that depending on the context and duration of exposure, GCs can increase some of the inflammatory responses they normally inhibit in central nervous system (CNS) [
13‐
15]. Chronic GC exposure that would normally suppress inflammatory responses in the periphery instead lead to increased CNS inflammation in response to bacterial lipopolysaccharide (LPS) [
16] and excitotoxin, particularly in GR-rich regions like the frontal cortex and hippocampus [
17]. Several studies have also shown that chronic stress and GCs can modulate the immunophenotype of CNS macrophages and microglia [
14,
18,
19], augment the microglial proinflammatory response to LPS [
20,
21], and enhance the TNF-α-mediated increase of Toll-like receptor (TLR) [
22]. These data demonstrate that chronic exposure to GCs primes microglia to proinflammatory stimuli and suggest that GCs may have proinflammatory action. However, it remains unclear whether chronic GC exposure has proinflammatory effects on hippocampal neurons.
Inflammasomes are multi-protein complexes that regulate the activity of caspase-1 and promote the maturation of inflammatory cytokines IL-1β and IL-18, which have been shown to play an important role in neuronal injury [
23]. The nucleotide-binding oligomerization domain (NOD) like receptor protein 1 (NLRP1) inflammasome is the first to be discovered and is composed of NLRP-1, an adaptor known as apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC), and caspase-1 [
24]. NLRP1 inflammasome was mainly expressed in neurons and implicated in the processes of AD and epilepsy [
12,
25,
26]. It has been reported that chronic GC exposure increased the gene expression of NLRP3, Iba-1, MHCII, and NF-κBIα in a concentration-dependent manner in microglia [
21]. Our latest study showed that chronic dexamethasone (DEX) treatment (21 and 28 days) induced significant neurodegeneration and activated NLRP1 inflammasome in the frontal cortex and hippocampus brain tissue [
27]. However, the precise mechanisms of chronic GC exposure on the activation of NLRP1 inflammasome in hippocampal neurons remain to be fully elucidated.
Recently, the role of K
+ in the activation of inflammasome is documented. Low intracellular K
+ concentration ([K
+]
i) is a requirement for NLRP1 and NLRP3 inflammasome activation [
28,
29]. In vitro, NLRP inflammasome assembly and caspase-1 recruitment occur spontaneously at [K
+]
i below 90 mM, but is prevented at higher concentrations [
30]. To induce NLRP3 activation, this mechanism of K
+ ions depletion additionally requires an influx of Ca
2+ through transient receptor potential (TRP) channels and activation of the TGF-β-activated kinase 1 (TAK1) [
28]. Large-conductance Ca
2+ and voltage-activated K
+ channels (BK channels), which are gated by Ca
2+ influx, contribute to action potential repolarization in neurons and play an important role in regulating neurotransmitter release [
31]. Outward K
+ currents through BK channels repolarize the cell and reduce excitability. Furthermore, BK channels are important for the K
+ transport [
32]. GCs have been shown to regulate BK channel sensitivity to phosphatase activity in pituitary-related cells. DEX, a synthetic glucocorticoid, reversibly increased the density BK current (
I
K(Ca)) in pituitary GH
3 and AtT-20 cells [
33]. However, to our knowledge, similar modulation of BK channels by GCs has not been shown in hippocampal neurons. And it is still unclear whether chronic GC exposure can induce the activation of NLRP1 inflammasome by regulating the BK channels.
DEX is a synthetic GC drug. The doses of DEX from 0.035 to 1 mg/kg were widely prescribed in clinic for treating many diseases [
34,
35], while the doses from 0.5 to 80 mg/kg were widely used in animals to study the neurodegenerative diseases [
36‐
38]. Our prior study showed that DEX (5 μM) exposure for 3 days significantly increased expression of NLRP1 inflammasome in hippocampal neurons [
39]. In the present study, we further investigated the mechanisms of chronic DEX (5 μM) treatment on BK-NLRP1 inflammasome signal in hippocampal neurons. The study had the potential to contribute to a more complete understanding of the mechanisms by which GCs may involve in neurodegeneration and progression of AD.
Discussion
Chronic stress has been reported to be associated with many neurodegenerative diseases, such as depression, AD, and PD [
2,
4,
49]. The chronic stress-induced neurodegenerative diseases are an outcome of different mechanisms, such as central neurotransmitters, neurohormonal factors, free radical generation, particularly the dysfunction of hypothalamic-pituitary-adrenal (HPA) axis [
50,
51]. GCs are the primary hormones released from the adrenal gland in response to stressful events. It has been reported that the physiological plasma corticosterone concentration range in rats is roughly between 20 and 50 nM, while the stress levels of this hormone are considered to be from 100 to 200 nM or even higher [
52]. Growing data suggest that high level of plasma GCs may be an important cause of chronic stress-induced neurodegeneration. In prior studies, hippocampal microglia isolated from chronic GC-exposed animals showed a potentiated response to LPS, which demonstrates that chronic GC exposure primes microglia to pro-inflammatory stimuli [
18,
21]. Our prior study showed that chronic DEX exposure significantly activated the NLRP1 inflammasome and induced neuronal damage in hippocampal neurons [
39]. In the current study, we demonstrate that NLRP1 inflammasome is activated by chronic DEX treatment and BK channel K
+ signal mediates chronic DEX exposure-induced NLRP1 inflammasome activation, which is accountable for chronic GCs induced hippocampal neurons injury.
It has been reported that low [K
+]
i (below 90 mM) could activate NLRP1 inflammasome in immune cells [
30]. Also, valinomycin-triggered K
+ efflux activates caspase-1 and increases IL-1β secretion in cultured spinal cord neurons [
53]. Thus, [K
+]
i may be a critical element in the activation of NLRP1 inflammasome. BK channels, which are gated by Ca
2+ and voltage, contribute to action potential repolarization in neurons and play an important role in regulating [K
+]
i [
32]. GCs have been shown to regulate BK channel sensitivity to phosphatase activity in pituitary-related cells. Dexamethasone reversibly increases the density of BK current in pituitary GH
3 and AtT-20 cells [
33]. At present, whether chronic GC exposure can mediate NLRP1 inflammasome activation by increasing BK channel function is still unclear. We hypothesize that chronic GC exposure may upregulate the expression of BK channel, increase K
+ efflux, and lead to low [K
+]
i, which mediates the activation of NLRP1 inflammasome and induces hippocampal neurons injury.
To confirm our hypothesis, we first investigated the effects of chronic DEX and BK channel inhibitor IbTx treatment on hippocampal neurons injury in vitro. We found that DEX treatment significantly increased LDH release in supernatant and accelerated hippocampal neuron apoptosis, while DEX failed to increase LDH release in the presence of IbTx. The results suggest that the hippocampal neuron injury induced by chronic DEX exposure is sensitive to IbTx. Meanwhile, we found that IbTx had a trend to decrease neuronal apoptosis (
P > 0.05). It is unclear what is responsible for the phenomenon. We think that chronic DEX exposure may lead to neuronal damage and apoptosis and IbTx may mainly inhibit the neuronal damage, such as inflammatory injury, rather than apoptosis. Further efforts will be made to clarify the precise mechanism in future research. MAP2, a cytoskeletal protein localized in the neuronal dendritic compartment, is considered a marker of structural integrity because it is involved in morphological stabilization of dendritic processes [
46]. The expression of MAP2 coincides with dendritic outgrowth, branching, and postlesion dendritic remodeling, suggesting that MAP2 plays a crucial role in plasticity of neurons [
54]. In the present study, we found that chronic DEX treatment for 5 days significantly decreased the expression of MAP2 in hippocampal neurons. IbTx could increase the expression of MAP2 which reduced by chronic DEX treatment. These data suggest that chronic GC exposure can induce hippocampal neurons injury and the mechanism may be related to the regulation of BK-NLRP1 inflammasome signal.
The inflammasomes are multiprotein complexes that are responsible for the formation of proinflammatory molecules. The NLRP1 inflammasome is first characterized as a member of the NLRP family, whose activation can generate a functional caspase-1-containing inflammasome to cleave the precursors of IL-1β and IL-18 to yield active cytokines [
26]. NLRP1 inflammasome is also highly expressed in pyramidal neurons of the brain [
55] and has a key role in the pathogenesis of neurological disorders [
12,
56]. The NLRP1 inflammasome consists of NLRP1, ASC, and caspase-1 [
57]. The ASC is an important component of the inflammasomes, which connects the NLRPs to caspase-1 [
58]. Caspase-1 is a critical modulator for maturation from pro-IL-1β and pro-IL-18 to their biologically active forms of IL-1β and IL-18 [
59]. Therefore, the inflammasome is necessary for caspase-1 activation and IL-1β and IL-18 release and participates in the amplification of the inflammatory response and the promotion of cell death [
60,
61]. Our earlier results showed that DEX 5 μM treatment for 3 days significantly activated NLRP1 inflammasome and increase the release of IL-1β and IL-18 in the supernatant. GC receptor antagonist RU486 could significantly decrease the expression of NLRP1, caspase-1, and IL-1β in hippocampal neurons and reduce the release of IL-1β and IL-18 [
39]. However, whether GCs can activate the NLRP1 inflammasome by modulating BK channels remains unknown. In the present study, the results showed that DEX treatment for 3 days significantly increased the release of IL-1β and IL-18 in supernatant and increased the expression of NLRP1, ASC, caspase-1, and IL-1β in hippocampal neurons, while IbTx could inhibit DEX-induced activation of NLRP1 inflammasome in hippocampal neurons. These results suggest that chronic GC exposure can induce NLRP1 inflammasome activation and BK channel may be involved in regulation of NLRP1 inflammasome induced by chronic DEX treatment.
The BK channel is ubiquitously expressed in the nervous system and plays an important modulator of neuronal function. It has been reported that BK channel could modulate neuronal excitability, firing rate, and neurotransmitter release [
62‐
64]. The ability of GCs to both reduce neuronal firing rate in celiac ganglion cells and enhance firing rate in cardiovascular neurons located in the rostral ventrolateral medulla [
65,
66] shows the importance of rapid steroid modulation in neuronal excitability. Recently, the acute application of DEX has been shown to increase BK channel activity in pituitary GH3 and AtT-20 cells and reduce the firing of action potentials in GH3 cells [
33]. Moreover, GCs could facilitate BK activation in adrenal chromaffin cells and promoting rapid action potential repolarization [
67]. Similar effects of GCs on pituitary corticotrope and somatotrope like cell lines have also been reported [
33]. However, modulation of BK channels by GCs in hippocampal neurons has not been fully elucidated. Whether GCs modulate BK channels and involve in NLRP1 inflammasome in hippocampal neurons is not yet known.
Low [K
+]
i is a potent activator for the NALP1 inflammasome, which then stimulates caspase-1 to cleave the proforms of IL-1ß and IL-18 cytokines [
29]. Our prior study showed that DEX (5 mg/kg) treatment for 28 days significantly increased the expression of NLRP1 inflammasome and induced hippocampal neuronal damage [
27]. To confirm whether BK channels involve in chronic DEX exposure induced NLRP1 inflammasome activation, we further detected the effects of chronic DEX treatment on expression of BK channel in vivo and in vitro. The results showed that DEX (5 mg/kg) treatment for 28 days significantly increased the expression of BK mRNA and protein in hippocampus tissue in mice. Meanwhile, we found that DEX (5 μM) treatment for 3 days significantly increased the expression of BK channels, but failed to increase the BK channels expression in the presence of IbTx (0.2 μM) in hippocampal neurons. The results suggest that chronic DEX can upregulate expression of BK channel via gene effects and may be involved in activation of NLRP1 inflammasome in hippocampal neurons. It is still unknown whether changes in BK activity are correlated with changes in [K
+]
i. To confirm whether DEX exposure can lead to low [K
+]
i by activating BK channel in hippocampal neurons, we detected the acute effect of DEX and IbTx treatment for 2 h on [K
+]
i in hippocampal neurons in vitro. We found that DEX treatment for 2 h significantly decreased [K
+]
i in hippocampal neurons. The BK channel inhibitor IbTx could significantly increase [K
+]
i in hippocampal neurons. The results suggest that DEX may decrease [K
+]
i by activating BK channel via non gene effects.
Furthermore, it has been reported that physiologically relevant concentrations of GCs facilitate gating of BK channels in HEK-293 cells, within 10 s of application to cell-free inside-out patches and under whole cell conditions [
68]. Therefore, we proposed that DEX might increase BK channel currents, which contribute to the lower [K
+]
i and the activation of NLRP1 inflammasome in hippocampal neurons. We further detected the acute effect of DEX incubation for 5 min on BK channel currents in hippocampal neurons. The results showed that extracellular DEX (1, 5 μM) treatment significantly increased the BK channel currents and IbTx, the BK channel inhibitor, significantly reduced the effect. These data suggest that GC acute exposure (5 min) can activate the BK channel, which may involve in the lower [K
+]
i induced by DEX sustained exposure (2 h).