Activation of LXRs alleviates neuropathic pain-induced cognitive dysfunction by modulation of microglia polarization and synaptic plasticity via PI3K/AKT pathway.

OBJECTIVE: Cognitive dysfunction is a common comorbidity in patients with chronic pain. Activation of Liver X receptors (LXRs) plays a potential role in improving cognitive disorders in central nervous diseases. In this study, we investigated the role of LXRs in cognitive deficits induced by neuropathic pain. METHODS: We established the spared nerve injury (SNI) model to investigate pain-induced memory dysfunction. Pharmacological activation of LXRs with T0901317 or inhibition with GSK2033 was applied. PI3K inhibitor LY294002 was administered to explore the underlying mechanism of LXRs. Changes in neuroinflammation, microglia polarization, and synaptic plasticity were assessed using biochemical technologies. RESULTS: We found that SNI-induced cognitive impairment was associated with reduced LXRß expression, increased M1-phenotype microglia, decreased synaptic proteins, and inhibition of PI3K/AKT signaling pathway in the hippocampus. Activation of LXRs using T0901317 effectively alleviated SNI-induced cognitive impairment. Additionally, T0901317 promoted the polarization of microglia from M1 to M2, reduced pro-inflammatory cytokines, and upregulated synaptic proteins in the hippocampus. However, administration of GSK2033 or LY294002 abolished these protective effects of T0901317 in SNI mice. CONCLUSIONS: LXRs activation alleviates neuropathic pain-induced cognitive impairment by modulating microglia polarization, neuroinflammation, and synaptic plasticity, at least partly via activation of PI3K/AKT signaling in the hippocampus. LXRs may be promising targets for addressing pain-related cognitive deficits.

Rapid injection of lumbar dorsal root ganglia under direct vision: Relevant anatomy, protocol, and behaviors.

Introduction: Dorsal root ganglia (DRG) are anatomically well-defined structures that contain all primary sensory neurons and are distension nodules of the dorsal root in the spinal cord near the medial surface of each foramen. Therefore, DRG is considered to be a desirable target for injection to manage chronic pain. But it presents a limitation in probing deep into it without in vivo injection technology. Methods: Here, we described a technique for administering intraganglionic injections of lumbar DRG under direct vision. We use partial osteotomy rather than laminectomy, which removes more bone, to preserve spinal structures while gaining adequate DRG access. To monitor the intraoperative progress of the DRG injection, a non-toxic dye was utilized. The effectiveness of the injection on the diffusion of AAV (adeno-associated virus) within the ganglion was assessed by histopathology at postoperative day 21. Results: Behavioral tests showed that neither motor nor sensory abilities were affected by saline or AAV injections. Meanwhile, the decreased pain threshold of SNI (spared nerve injury) was considerably restored by pharmacological inhibition of DRG neurons. Discussion: Our research achieved a new minimally invasive and intuitive intra-ganglionic injection in mice. In addition, the present protocol may serve as a valuable resource for planning preclinical studies of DRG injection.

Celastrol inhibits store operated calcium entry and suppresses psoriasis.

Introduction: Psoriasis is an inflammatory autoimmune skin disease that is hard to cure and prone to relapse. Currently available global immunosuppressive agents for psoriasis may cause severe side effects, thus it is crucial to identify new therapeutic reagents and druggable signaling pathways for psoriasis. Methods: To check the effects of SOCE inhibitors on psoriasis, we used animal models, biochemical approaches, together with various imaging techniques, including calcium, confocal and FRET imaging. Results and discussion: Store operated calcium (Ca2+) entry (SOCE), mediated by STIM1 and Orai1, is crucial for the function of keratinocytes and immune cells, the two major players in psoriasis. Here we showed that a natural compound celastrol is a novel SOCE inhibitor, and it ameliorated the skin lesion and reduced PASI scores in imiquimod-induced psoriasis-like mice. Celastrol dose- and time-dependently inhibited SOCE in HEK cells and HaCaT cells, a keratinocyte cell line. Mechanistically, celastrol inhibited SOCE via its actions both on STIM1 and Orai1. It inhibited Ca2+ entry through constitutively-active Orai1 mutants independent of STIM1. Rather than blocking the conformational switch and oligomerization of STIM1 during SOCE activation, celastrol diminished the transition from oligomerized STIM1 into aggregates, thus locking STIM1 in a partially active state. As a result, it abolished the functional coupling between STIM1 and Orai1, diminishing SOCE signals. Overall, our findings identified a new SOCE inhibitor celastrol that suppresses psoriasis, suggesting that SOCE pathway may serve as a new druggable target for treating psoriasis.

Spinal voltage-gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome.

BACKGROUD: Studies have shown that the activation of microglia is the main mechanism of neuropathic pain. Kv1.3 channel is a novel therapeutic target for treating neuroinflammatory disorders due to its crucial role in subsets of microglial cells. As such, it may be involved in the processes of neuropathic pain, however, whether Kv1.3 plays a role in neuroinflammation following peripheral nerve injury is unclear. METHOD: The spared nerve injury model (SNI) was used to establish neuropathic pain. Western blot and immunofluorescence were used to examine the effect of Kv1.3 in the SNI rats. PAP-1, a Kv1.3 specific blocker was administered to alleviate neuropathic pain in the SNI rats. RESULTS: Neuropathic pain and allodynia occurred after SNI, the levels of M1 (CD68, iNos) and M2 (CD206, Arg-1) phenotypes were up-regulated in the spinal cord, and the protein levels of NLRP3, caspase-1 and IL-1ß were also increased. Pharmacological blocking of Kv1.3 with PAP-1 alleviated hyperpathia induced by SNI. Meanwhile, intrathecal injection of PAP-1 reduced M1 polarization and decreased NLRP3, caspase-1 and IL-1ß expressions of protein levels. CONCLUSION: Our research indicates that the Kv1.3 channel in the spinal cord contributes to neuropathic pain by promoting microglial M1 polarization and activating the NLRP3 inflammasome.

Dysfunction of the Brain-derived Neurotrophic Factor-Tyrosine Kinase B Signaling Pathway Contributes to Learning and Memory Impairments Induced by Neuroinflammation in Mice.

Accumulating evidence suggests that neuroinflammation is the main mechanism in cognitive dysfunction and that brain-derived neurotrophic factor (BDNF) is involved in learning and memory by binding to tyrosine kinase B (TrkB) receptors. Herein, we tested the roles of the BDNF-TrkB signaling pathway and its downstream cascade in lipopolysaccharide (LPS) induced cognitive dysfunction in mice. Mice were treated with LPS (0.25 mg/kg) for 7 days, and learning and memory function was evaluated by the novel object recognition test (NORT). Western blotting was performed to elucidate roles of the BDNF-TrkB signaling pathway and its downstream cascades in LPS mice. The NORT showed that LPS induced learning and memory deficits in mice. The levels of IL-1ß, IL-6, and TNF-α in the serum and central nervous system decreased in LPS mice. In addition, LPS reduced the protein levels of BDNF, p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 and increased the expression of Bax in the hippocampus and medial prefrontal cortex regions. In the entorhinal cortex, the protein levels of BDNF, p-TrkB, Bcl-2, p-CaMK2 and p-CREB were decreased, and the protein level of Bax was increased in LPS mice. Interestingly, 7,8-DHF alleviated these disorders in LPS mice and improved learning and memory function; however, the TrkB antagonist ANA12 effectively reversed effects of 7,8-DHF. Therefore, we conclude that the BDNF-TrkB signaling pathway and its downstream cascades disorders in different regions are main mechanisms of cognitive dysfunction, and 7,8-DHF maybe useful as a new treatment for preventing or treating cognitive dysfunction induced by neuroinflammation in neurodegenerative diseases.

Deficiency of ER Ca2+ sensor STIM1 in AgRP neurons confers protection against dietary obesity.

Store-operated calcium entry (SOCE) is pivotal in maintaining intracellular Ca2+ level and cell function; however, its role in obesity development remains largely unknown. Here, we show that the stromal interaction molecule 1 (Stim1), an endoplasmic reticulum (ER) Ca2+ sensor for SOCE, is critically involved in obesity development. Pharmacological blockade of SOCE in the brain, or disruption of Stim1 in hypothalamic agouti-related peptide (AgRP)-producing neurons (ASKO), significantly ameliorates dietary obesity and its associated metabolic disorders. Conversely, constitutive activation of Stim1 in AgRP neurons leads to an obesity-like phenotype. We show that the blockade of SOCE suppresses general translation in neuronal cells via the 2',5'-oligoadenylate synthetase 3 (Oas3)-RNase L signaling. While Oas3 overexpression in AgRP neurons protects mice against dietary obesity, deactivation of RNase L in these neurons significantly abolishes the effect of ASKO. These findings highlight an important role of Stim1 and SOCE in the development of obesity.

The Role of the GABAergic System in Diseases of the Central Nervous System.

It is well known that the central nervous system (CNS) is a complex neuronal network and its function depends on the balance between excitatory and inhibitory neurons. Disruption of the excitatory/inhibitory (E/I) balance is the main cause for the majority of the CNS diseases. In this review, we will discuss roles of the inhibitory system in the CNS diseases. The GABAergic system as the main inhibitory system, is essential for the appropriate functioning of the CNS, especially as it is engaged in the formation of learning and memory. Many researchers have reported that the GABAergic system is involved in regulating synaptic plasticity, cognition and long-term potentiation. Some clinical manifestations (such as cognitive dysfunctions, attention deficits, etc.) have also been shown to emerge after abnormalities in the GABAergic system accompanied with concomitant diseases, that include Alzheimer's disease (AD), Parkinson's disease (PD), Autism spectrum disorder (ASD), Schizophrenia, etc. The GABAergic system consists of GABA, GABA transporters, GABAergic receptors and GABAergic neurons. Changes in any of these components may contribute to the dysfunctions of the CNS. In this review, we will synthesize studies which demonstrate how the GABAergic system participates in the pathogenesis of the CNS disorders, which may provide a new idea that might be used to treat the CNS diseases.

GLP-1R activation ameliorated novel-object recognition memory dysfunction via regulating hippocampal AMPK/NF-κB pathway in neuropathic pain mice.

Growing evidences indicate that neuropathic pain is frequently accompanied with cognitive impairments, which aggravate the decrease in the quality of life of chronic pain patients. Furthermore, it has been shown that the activation of Glucagon-like-peptide-1receptor (GLP-1R) improved memory deficit in multiple diseases, including Alzheimer's disease (AD), stroke. However, whether GLP-1R activation could improve memory impairment induced by neuropathic pain and the mechanisms underlying the effect of the activation of GLP-1R on memory protection have not yet been established. The spared nerve injury (SNI) model was established as a kind of neuropathic pain. And novel-object recognition memory (hippocampus-dependent memory) was tested by the novel object recognition test (NORT). The expression levels of GLP-1, GLP-1R, adenosine monophosphate-activated protein kinase (AMPK), p-AMPKThr172, nuclear factor κ B p65 (NF-κB p65), interleukin-1beta (IL-1ß), IL-1ß p17 (mature IL-1ß), tumor necrosis factor-alpha (TNF-α) and the synaptic proteins were tested in the murine hippocampus with memory deficits caused by neuropathic pain. Then, exenatide acetate (Ex-4, a GLP-1R agonist), exendin (9-39) (Ex(9-39), a GLP-1R antagonist) and Compound C dihydrochloride (CC, an AMPK inhibitor) were used to test the effects of the activation of GLP-1R in the mice with neuropathic pain. First, we uncovered that neuropathic pain could inhibit GLP-1/GLP-R axis, disturb inflammatory signaling pathway, increase the expression of IL-1ß, IL-1ß p17 and TNF-α, downregulate the synaptic proteins (postsynaptic density protein 95 (PSD95) and Arc). Subsequently, we reported that Ex-4 treatment could improve recognition memory impairment, increase the ratio of p-AMPKThr172/AMPK, inhibit the phosphorylation NF-κB p65 and decrease the expression of IL-1ß, IL-1ß p17 and TNF-α, upregulate the levels of PSD95 and Arc. Moreover, we found that Ex(9-39) and CC treatment could abrogate the memory protection of activation of GLP-1R in mice with neuropathic pain. The results indicated that the activation of GLP-1R could improve recognition memory impairment via regulating AMPK/NF-κB pathway, improving neuroinflammation, reversing the decreased level of synaptic proteins in neuropathic pain mice.

Identification of molecular determinants that govern distinct STIM2 activation dynamics.

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.