Enhancement of the SESN2-SHP cascade by melatonin ameliorates hepatic gluconeogenesis by inhibiting the CRBN-BTG2-CREBH signaling pathway.

Melatonin is involved in the regulation of various biological functions. Here, we explored a novel molecular mechanism by which the melatonin-induced sestrin2 (SESN2)-small heterodimer partner (SHP) signaling pathway protects against fasting- and diabetes-mediated hepatic glucose metabolism. Various key gene expression analyses were performed and multiple metabolic changes were assessed in liver specimens and primary hepatocytes of mice and human participants. The expression of the hepatic cereblon (CRBN) and b-cell translocation gene 2 (BTG2) genes was significantly increased in fasting mice, diabetic mice, and patients with diabetes. Overexpression of Crbn and Btg2 increased hepatic gluconeogenesis by enhancing cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CREBH), whereas this phenomenon was prominently ablated in Crbn null mice and Btg2-silenced mice. Interestingly, melatonin-induced SESN2 and SHP markedly reduced hepatic glucose metabolism in diabetic mice and primary hepatocytes, and this protective effect of melatonin was strikingly reversed by silencing Sesn2 and Shp. Finally, the melatonin-induced SESN2-SHP signaling pathway inhibited CRBN- and BTG2-mediated hepatic gluconeogenic gene transcription via the competition of BTG2 and the interaction of CREBH. Mitigation of the CRBN-BTG2-CREBH axis by the melatonin-SESN2-SHP signaling network may provide a novel therapeutic strategy to treat metabolic dysfunction due to diabetes.

Comparative analysis of dermal collagen and lipids in cereblon ablated mice using a multimodal nonlinear optical system.

By utilizing a multimodal nonlinear optical system that combines coherent anti-Stokes Raman scattering and second harmonic generation to investigate biological characteristics of dermal tissues ex vivo, we demonstrate the potential feasibility of using this optical approach as a powerful new investigative tool for future biomedical research. For this study, our optical system was utilized for the first time to analyze lipid and collagen profiles in cereblon knockout (KO) mouse skin, and we were able to discover significant alterations in the number of carbon-carbon double bonds (wild-type vs. cereblon KO; NCC : 0.75 vs. 0.85) of skin fatty acids in triacylglycerides as well as changes in dermal collagen fibers (25% reduction in cereblon KO). By adopting our optical system to biological studies, we provide researchers with another diagnostic approach to validate their experimental results, which will significantly advance the state of biomedical research.

Effects of cereblon on stress-activated redox proteins and core behavior.

The mechanisms underlying the vulnerability and resilience of an individual to stress are only partly understood. Response to stress is determined by behavioral and biochemical changes in the brain. Chronic ultra-mild stress (CUMS) induces an anhedonic-like state in mice that resembles symptoms of human depression. This study reports the role of cereblon (CRBN) in regulating the metabolic and antioxidant status of neuronal tissues in the mouse model of CUMS. Intriguingly, Crbn-/- (KO) mice showed resilient responsiveness, both at the behavioral and proteomic levels. Several core behaviors were also differentially altered by CUMS in KO mice. Liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based proteome analysis of whole brain lysate (WBL) showed an enriched chaperonic, metabolic, and antioxidant status in the brains of KO subjects, including several members of DNAJ chaperones, creatine kinase, quinone oxidoreductase, superoxide dismutase (SOD1), glutathione S-transferase Mu (GSTM), peroxiredoxin-6 (PRDX6), and thioredoxin. Pathological phosphorylation as characterized by aggregation of tau and α-synuclein (α-syn) was significantly reduced in the neuronal tissues of KO mouse model of CUMS as compared to wild type (WT) mice. Furthermore, significantly increased SOD1 activity and reduced lipid peroxidation were observed in Crbn-KO systems. Integrated signaling pathways were also identified in CRBN-specific sub-networks constructed from protein-protein interaction analysis by STRING. The present study highlights the roles of CRBN in regulating the stress response (SR) and reshaping metabolic status in the brains of mice exposed to CUMS. A better understanding of the molecular mechanisms of depression and neurodegeneration can improve the development of novel treatments.

Novel Thioxothiazolo[3,4-a]quinazolin-5(4H)-one Derivatives as BKCa Channel Activators for Urinary Incontinence.

Overactive bladder (OAB) is a syndrome causing a sudden and unstoppable need to urinate with significant global prevalence. Several drugs are used to treat OAB; however, they have various side effects. Therefore, new treatment options for OAB are required. A series of novel 5-oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide derivatives were synthesized and evaluated for their large-conductance voltage- and Ca2+-activated K+ channel activation through a cell-based fluorescence assay and electrophysiological recordings. Several compounds, including a 7-bromo substituent on the heterocyclic system, showed increased channel currents. Among the derivatives, compound 12h exhibited potent in vitro activity with a half-maximal effective concentration (EC50) of 2.89 µM, good oral pharmacokinetic properties (area under the curve and half-life), and in vivo efficacy in a spontaneously hypertensive rat model.

Discovery and characterization of a potent activator of the BKCa channel that relives overactive bladder syndrome in rats.

The large-conductance Ca2+-activated K+ channel (BKCa channel) is involved in repolarizing the membrane potential and has a variety of cellular functions. The BKCa channel is highly expressed in bladder smooth muscle and mediates muscle relaxation. Compounds that activate the BKCa channel have therapeutic potential against pathological symptoms associated with the overactivity of bladder smooth muscle. In this regard, we screened a chemical library of 9938 compounds to identify novel BKCa channel activators. A cell-based fluorescence assay identified a structural family of compounds containing a common tricyclic quinazoline ring that activated the BKCa channel. The most potent compound TTQC-1 (7-bromo-N-(3-methylphenyl)-5-oxo-1-thioxo-4,5-dihydro[1,3]thiazolo[3,4-a]quinazoline-3-carboxamide) directly and reversibly activated the macroscopic current of BKCa channels expressed in Xenopus oocytes from both sides of the cellular membrane. TTQC-1 increased the maximum conductance and shifted the half activation voltage to the left. The apparent half-maximal effective concentration and dissociation constant were 2.8 µM and 7.95 µM, respectively. TTQC-1 delayed the kinetics of channel deactivation without affecting channel activation. The activation effects were observed over a wide range of intracellular Ca2+ concentrations and dependent on the co-expression of ß1 and ß4 auxiliary subunits, which are highly expressed in urinary bladder. In the isolated smooth muscle cells of rat urinary bladder, TTQC-1 increased the K+ currents which can be blocked by iberiotoxin. Finally, oral administration of TTQC-1 to hypertensive rats decreased the urination frequency. Therefore, TTQC-1 is a BKCa channel activator with a novel structure that is a potential therapeutic candidate for BKCa channel-related diseases, such as overactive bladder syndrome.

Cereblon contributes to cardiac dysfunction by degrading Cav1.2α.

AIMS: Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. METHODS AND RESULTS: Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. CONCLUSION: Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics.

Cereblon: promise and challenges for combating human diseases.

Cereblon (CRBN) is a substrate recognition protein in the E3-ligase ubiquitin complex. The binding target of CRBN varies according to tissues and cells, and the protein regulates various biological functions by regulating tissue-specific targets. As new endogenous targets of CRBN have been identified over the past decade, the physiological and pathological functions of CRBN and its potential as a therapeutic target in various diseases have greatly expanded. For this purpose, in this review article, we introduce the basic principle of the ubiquitin-proteasome system, the regulation of physiological/pathological functions related to the endogenous substrate of CRBN, and the discovery of immunomodulatory imide drug-mediated neo-substrates of CRBN. In addition, the development of CRBN-based proteolysis-targeting chimeras, which has been actively researched recently, and strategies for developing therapeutic agents using them are introduced. These recent updates on CRBN will be useful in the establishment of strategies for disease treatment and utilization of CRBNs in biomedical engineering and clinical medicine.

Regulation of AMPK Activity by CRBN Is Independent of the Thalidomide-CRL4CRBN Protein Degradation Axis.

Cereblon (CRBN), a primary target of immune-modulatory imide drugs (IMiDs), functions as a substrate receptor in the CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) E3 ubiquitin ligase complex. Binding of IMiDs to CRBN redirects the CRL4CRBN E3 ubiquitin ligase to recruit or displace its substrates. Interaction between CRBN and the AMPK α subunit leads to CRL4CRBN-dependent degradation of the γ subunit and inhibits AMPK activity. However, the effect of thalidomide on the function of CRBN as a negative regulator of AMPK through interaction with the α subunit remains unclear. Here, we show that thalidomide does not affect AMPK activation or the binding affinity between CRBN and the AMPK α subunit. Thalidomide had no effect on AMPK activity independent of CRBN expression. The N-terminal region and C-terminal tail of CRBN, which is distinct from the IMiD binding site, were critical for interaction with the AMPK α subunit. The present results suggest that CRL4CRBN negatively regulates AMPK through a pathway independent from the CRBN-IMiD binding region.

Synthesis and BK channel-opening activity of 2-amino-1,3-thiazole derivatives.

A series of 2-amino-5-arylmethyl- or 5-heteroarylmethyl-1,3-thiazole derivatives were synthesized and evaluated for BK channel-opening activities in cell-based fluorescence assay and electrophysiological recording. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at benzene ring.

Cereblon Regulates the Proteotoxicity of Tau by Tuning the Chaperone Activity of DNAJA1.

Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/ß, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that Crbn-/- mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.

Ablation of CRBN induces loss of type I collagen and SCH in mouse skin by fibroblast senescence via the p38 MAPK pathway.

Cereblon (CRBN) is a substrate receptor of the cullin-RING E3 ubiquitin ligase (CRL) complex that mediates the ubiquitination of several substrates. In this study, CRBN knockout (KO) mice exhibited decreased levels of stratum corneum hydration (SCH) and collagen I expression with an elevated protein level of matrix metalloprotease 1 (MMP1). The absence of cereblon in the skin of CRBN KO mice mimics the damage caused by narrowband ultraviolet B (NB-UVB). The primary CRBN deficient mouse embryonic fibroblasts (MEFs) undergo G2/M-arrested premature senescence via protein signaling of p38 MAPK and its dependent p53/p21pathway. The absence of CRBN induced the markers of cellular senescence, such as the senescence-associated heterochromatin foci (SAHF), SA-ß-Gal staining, and p21 upregulation while the ectopic expression of CRBN reversed the phenotypes of SA-ß-Gal staining and p21 upregulation. Reversion of the decreased protein level of collagen I was demonstrated after the reintroduction of the CRBN gene back into CRBN KO MEFs, validating the promising role of CRBN as a potential regulator for the function of the skin barrier and its cellular homeostasis.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

Crbn modulates calcium influx by regulating Orai1 during efferocytosis.

Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. However, the molecular mechanism by which calcium flux is modulated during efferocytosis remains elusive. Here, we report that Orai1, a Crbn substrate, is upregulated via its attenuated interaction with Crbn during efferocytosis, which increases calcium influx into phagocytes and thereby promotes efferocytosis. We found that Crbn deficiency promoted phagocytosis of apoptotic cells, which resulted from facilitated phagocytic cup closure and was nullified by a CRAC channel inhibitor. In addition, Orai1 associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai1 and alteration of SOCE-mediated calcium influx. The association of Orai1 with Crbn was attenuated during efferocytosis, leading to reduced ubiquitination of Orai1 and consequently upregulation of Orai1 and calcium influx. Collectively, our study reveals a regulatory mechanism by which calcium influx is modulated by a Crbn-Orai1 axis to facilitate efferocytosis.

Susceptibility of pentylenetetrazole-induced seizures in mice with Cereblon gene knockout.

Epilepsy is a neurological disorder characterized by unpredictable seizures, which are bursts of electrical activity that temporarily affect the brain. Cereblon (CRBN), a DCAFs (DDB1 and CUL4-associated factors), is a well-established protein associated with human mental retardation. Being a substrate receptor of the cullin-RING E3 ubiquitin ligase (CRL) 4 complex, CRBN mediates ubiquitination of several substrates and conducts multiple biological processes. In the central nervous system, the largeconductance Ca2+-activated K+ (BKCa) channel, which is the substrate of CRBN, is an important regulator of epilepsy. Despite the functional role and importance of CRBN in the brain, direct injection of pentylenetetrazole (PTZ) to induce seizures in CRBN knock-out mice has not been challenged. In this study, we investigated the effect of PTZ in CRBN knock-out mice. Here, we demonstrate that, compared with WT mice, CRBN knock-out mice do not show the intensification of seizures by PTZ induction. Moreover, electroencephalography recordings were also performed in the brains of both WT and CRBN knockout mice to identify the absence of significant differences in the pattern of seizure activities. Consistently, immunoblot analysis for validating the protein level of the CRL4 complex containing CRBN (CRL4Crbn) in the mouse brain was carried out. Taken together, we found that the deficiency of CRBN does not affect PTZ-induced seizure. [BMB Reports 2020; 53(9): 484-489].

Neurite growth of trigeminal ganglion neurons in vitro with near-infrared light irradiation.

Trigeminal ganglion (TG) neurons play an essential role in the sensory nerves of the face. Damaged TG neurons resulting from the accidental and non-intentional nerve lesions, commonly identified as neuropathic pain, which is known to cause intense pain and sensory abnormalities. For the treatment, surgical methods are conducted when the pharmacological treatment fails to provide satisfactory recovery. However, the process of surgery or drug intake can burden the patient or cause side effects. One of the logical choices of study becomes photobiomodulation (PBM) referred to as therapeutic approaches based on the interactions of visible or near-infrared (NIR) photons with biomolecules inside cells or tissues. In this study, we constructed a PBM illumination setup to stimulate the cultured primary TG neurons and compared the growth morphology between the non-irradiated control group and irradiation group with NIR laser of 808 nm wavelength. In addition, we applied various radiant exposures of 1, 2, and 10 J/cm2 with different pulse frequencies of 1, 10, and 100 Hz. We found that PBM could promote neurite growth of TG neurons, and it works at relatively low energy densities at 1 and 2 J/cm2. The irradiation group in the pulsed wave mode with the frequency of 10 Hz was found to be the most effective when compared to other frequencies. Thus, PBM on TG neurons facilitated neuronal growth in vitro in a dose and frequency-dependent fashion. PBM may provide a potential therapeutic approach to treat damaged peripheral nerves.

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity.

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn-/- mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn-/- mice. Increased stability of AMPKγ in Crbn-/- MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4CRBN directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4CRBN regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.

Induction of SIRT1 by melatonin improves alcohol-mediated oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway.

Alcoholic liver disease is the most prevalent chronic liver disease. Melatonin is known to control many vital processes. Here, we explored a novel molecular mechanism by which melatonin-induced SIRT1 signaling protects against alcohol-mediated oxidative stress and liver injury. Gene expression profiles and metabolic changes were measured in liver specimens of mice and human subjects. Expression levels of Cb1r, Crbn, Btg2, Yy1, pro-inflammatory cytokines, and Cyp2e1 were significantly enhanced in chronic alcohol-challenged mice and human subjects. Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic CYP2E1 protein, and reactive oxygen species (ROS) were elevated in alcohol-fed WT mice but not in Cb1r antagonist-treated, Crbn null, or Yy1-silenced mice. Importantly, alcohol-induced Yy1 and Cyp2e1 expression, ROS amount, and liver injury were markedly diminished by melatonin treatment and the transduction of Sirt1 in mice, whereas this phenomenon was prominently ablated by silencing of Sirt1. Notably, SIRT1 physically interacted with YY1 and attenuated YY1 occupancy on the Cyp2e1 gene promoter. Melatonin-SIRT1 signaling ameliorates alcohol-induced oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway. The manipulation of CRBN-YY1-CYP2E1 signaling network by the melatonin-SIRT1 pathway highlights a novel entry point for treating alcoholic liver disease.

Impairment of proprioceptive movement and mechanical nociception in Drosophila melanogaster larvae lacking Ppk30, a Drosophila member of the Degenerin/Epithelial Sodium Channel family.

The mechanosensory neurons of Drosophila larvae are demonstrably activated by diverse mechanical stimuli, but the mechanisms underlying this function are not completely understood. Here we report a genetic, immunohistochemical, and electrophysiological analysis of the Ppk30 ion channel, a member of the Drosophila pickpocket (ppk) family, counterpart of the mammalian Degenerin/Epithelial Na+ Channel family. Ppk30 mutant larvae displayed deficits in proprioceptive movement and mechanical nociception, which are detected by class IV sensory (mdIV) neurons. The same neurons also detect heat nociception, which was not impaired in ppk30 mutant larvae. Similarly, Ppk30 mutation did not alter gentle touch mechanosensation, a distinct mechanosensation detected by other neurons, suggesting that Ppk30 has a functional role in mechanosensation in mdIV neurons. Consistently, Ppk30 was expressed in class IV neurons, but was not detectable in other larval skin sensory neurons. Mutant phenotypes were rescued by expressing Ppk30 in mdIV neurons. Electrophysiological analysis of heterologous cells expressing Ppk30 did not detect mechanosensitive channel activities, but did detect acid-induced currents. These data show that Ppk30 has a role in mechanosensation, but not in thermosensation, in class IV neurons, and possibly has other functions related to acid response.

BK channel blocker paxilline attenuates thalidomide-caused synaptic and cognitive dysfunctions in mice.

Thalidomide is a widely prescribed immunomodulatory drug (iMiD) for multiple myeloma, but causes reversible memory loss in humans. However, how thalidomide causes cognitive dysfunction at a cellular and molecular level has not been demonstrated. We studied the effect of thalidomide on synaptic functions and cognitive behaviors using a mouse model. Thalidomide led to cognitive deficits in learning behavior in a passive avoidance test and in a novel object recognition test, increased anxiety in an elevated plus maze test, and increased depressive behaviors in a tail suspension test. Interestingly, thalidomide elevated big- or large-conductance, calcium-activated K+ (BK) channel expression in the plasma membrane and BK channel activity in the hippocampus. Thalidomide also increased the paired pulse ratio of excitatory postsynaptic current (EPSC), which suggests a decreased probability of glutamate release. Furthermore, the changes in the paired pulse ratio and in BK channel activity were blocked by paxilline, a BK channel blocker. Finally, we found that thalidomide-induced cognitive dysfunctions were restored by paxilline treatment. These results suggest that thalidomide-mediated BK channel hyperfunction is responsible for the pathological mechanism of thalidomide-associated reversible memory loss.