The cereblon-AMPK (AMP-activated protein kinase) axis in chondrocytes regulates the pathogenesis of osteoarthritis.

OBJECTIVE: AMP-activated protein kinase (AMPK) dysregulation is implicated in osteoarthritis (OA), but the mechanisms underlying this dysregulation remain unclear. We investigated the role of cereblon, a substrate-recognition protein within the E3-ligase ubiquitin complex, in AMPK dysregulation and OA pathogenesis. METHODS: Cereblon expression was examined in human (n = 5) and mouse (n = 10) OA cartilage. The role of cereblon was investigated through its adenoviral overexpression (n = 10) or knockout (KO, n = 15) in the destabilization of the medial meniscus (DMM)-operated mice. The therapeutic potentials of the chemical cereblon degrader, TD-165, and the AMPK activator, metformin, were assessed through intra-articular (IA) injection to mice (n = 15). RESULTS: Immunostaining revealed that cereblon is upregulated in human and mouse OA cartilage. In DMM model mice, cartilage destruction was exacerbated by overexpression of cereblon in mouse joint tissues (OARSI grade; 1.11 [95% CI: 0.50 to 2.75]), but inhibited in global (-2.50 [95% CI: -3.00 to -1.17]) and chondrocyte-specific (-2.17 [95% CI: -3.14 to -1.06]) cereblon KO mice. The inhibitory effects were more pronounced in mice fed a high-fat diet compared to a regular diet. The degradation of cereblon through IA injection of TD-165 inhibited OA cartilage destruction (-2.47 [95% CI: -3.22 to -1.56]). Mechanistically, cereblon exerts its catabolic effects by negatively modulating AMPK activity within chondrocytes. Consistently, activation of AMPK by IA injection of metformin inhibited posttraumatic OA cartilage destruction (-1.20 ([95% CI: -1.89 to -0.45]). CONCLUSIONS: The cereblon-AMPK axis acts as a catabolic regulator of OA pathogenesis and seems to be a promising therapeutic target in animal models of OA.

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.

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.

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.

Cereblon Maintains Synaptic and Cognitive Function by Regulating BK Channel.

Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. We investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models. Crbn knock-out (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). We found that the BK channel blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations.SIGNIFICANCE STATEMENTCereblon (CRBN), a well known target of the immunomodulatory drug thalidomide, was originally identified as a gene that causes human intellectual disability when mutated. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. Based on the idea that synaptic abnormalities are the most common factor in cognitive dysfunction, we monitored the synaptic structure and function of Crbn knock-out (KO) animals to identify the molecular mechanisms of intellectual disability. Here, we found that Crbn KO animals showed cognitive deficits caused by enhanced BK channel activity and reduced presynaptic glutamate release. Our findings suggest a physiological pathomechanism of the intellectual disability-related gene CRBN and will contribute to the development of therapeutic strategies for CRBN-related intellectual disability.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Cereblon in health and disease.

Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that has been linked to autosomal recessive non-syndromic mental retardation. Several key findings suggest diverse roles of CRBN, including its regulation of the large-conductance calcium- and voltage-activated potassium (BKCa) channels, regulation of thalidomide-binding proteins, and mediation of lenalidomide treatment in multiple myeloma. Recent studies also indicate that CRBN is involved in energy metabolism and negatively regulates AMP-activated protein kinase signaling. Mice with genetic depletion of CRBN are resistant to various stress conditions including a high-fat diet, endoplasmic reticulum stress, ischemia/reperfusion injury, and alcohol-related liver damage. In this review, we discuss the various roles of CRBN in human health and disease and suggest avenues for further research to enhance our basic knowledge and clinical application of CRBN.

Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

Functional effects of a pathogenic mutation in Cereblon (CRBN) on the regulation of protein synthesis via the AMPK-mTOR cascade.

Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK-mTOR cascade in Crbn-knock-out mice, ectopic expression of the wild-type CRBN increased protein synthesis by inhibiting endogenous AMPK. Unlike the wild-type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR-dependent repression of protein synthesis in Crbn-deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.

Ablation of cereblon attenuates myocardial ischemia-reperfusion injury.

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia-reperfusion (I-R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I-R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I-R (complete ligation of the coronary artery for 30 min followed by 24h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I-R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I-R injury through modulation of AMPK activity.

Effects of palmitoylation on the diffusional movement of BKCa channels in live cells.

BKCa channels are palmitoylated at a cluster of cysteine residues within the cytosolic linker connecting the 1st and 2nd transmembrane domains, and this lipid modification affects their surface expression. To verify the effects of palmitoylation on the diffusional dynamics of BKCa channels, we investigated their lateral movement. Compared to wild-type channels, the movement of mutant palmitoylation-deficient channels was much less confined and close to random. The diffusion of the mutant channel was also much faster than that of the wild type. Thus, the lateral movement of BKCa channels is greatly influenced by palmitoylation.

Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BK(Ca) channel modulators.

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BK(Ca)) channels is challenging because of the unique requirement of both voltage and high concentrations of Ca²âº for activation of these channels. Here, we describe a new cell-based assay system that utilizes a hyperactive mutant BK(Ca) channel. The hyperactive mutant was generated by introducing two point-mutations into the cytosolic flexible interface between the two RCK domains of the wild-type BK(Ca) channel. The mutant channel exhibited a large negative shift in its conductance-voltage relationship, which indicates activation by modest depolarization at resting concentrations of intracellular Ca²âº. Unlike the wild-type BK(Ca) channel, the hyperactive mutant did not require a concomitant increase of intracellular Ca²âº for activation. Despite the observed shift in its voltage activation profile, activity of the mutant channel was further potentiated by a known BK(Ca) channel activator. When tested in a commercially available cell-based K⁺ channel assay, cell-lines stably expressing the hyperactive BK(Ca) channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K⁺ channels. In summary, cell-lines expressing the hyperactive mutant BK(Ca) channel represent a new cell-based assay system for investigation of BK(Ca) channels that can be used to screen for novel modulators of these channels.

Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the ß7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the ß7 subunit.

Functional modulation of AMP-activated protein kinase by cereblon.

Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

Induction of cereblon by NF-E2-related factor 2 in neuroblastoma cells exposed to hypoxia-reoxygenation.

Cereblon is a protein encoded by the CRBN gene, which has been associated with human autosomal recessive nonsyndromic mental retardation. However, little is known about the regulation of CRBN expression. Following exposure of mouse neuroblastoma N2A cells to hypoxia/reoxygenation (H/R), mRNA and protein expression of CRBN were increased. To better understand how CRBN expression is regulated, the promoter region of the mouse CRBN gene was characterized functionally. Deletion mutations and site-directed mutagenesis led to the identification of a functional NF-E2-related factor 2 (Nrf2)-binding site. Electrophoretic mobility shift analysis indicated that Nrf2 binds to a putative binding site in the CRBN promoter. Nrf2 overexpression and tert-butylhydroquinone treatment enhanced CRBN protein expression. These results imply that Nrf2 stimulates CRBN gene transcription under H/R conditions in neuronal cells.

Regulation of dendritic spine morphology by SPIN90, a novel Shank binding partner.

Dendritic spines are highly specialized actin-rich structures on which the majority of excitatory synapses are formed in the mammalian CNS. SPIN90 is an actin-binding protein known to be highly enriched in postsynaptic densities (PSDs), though little is known about its function there. Here, we show that SPIN90 is a novel binding partner for Shank proteins in the PSD. SPIN90 and Shank co-immunoprecipitate from brain lysates and co-localize in postsynaptic dendrites and act synergistically to mediate spine maturation and spine head enlargement. At the same time, SPIN90 causes accumulation of Shank and PSD-95 within dendritic spines. In addition, we found that the protein composition of PSDs in SPIN90 knockout mice is altered as is the actin cytoskeleton of cultured hippocampal SPIN90 knockout neurons. Taken together, these findings demonstrate that SPIN90 is a Shank1b binding partner and a key contributor to the regulation of dendritic spine morphogenesis and brain function.

Establishment of an assay for P2X7 receptor-mediated cell death.

The P2X7 receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human P2X7 receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of hP2X7 receptor. Functional activity of the hP2X7 receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the hP2X7-expressing HEK 293 cells and this cell death could be quantified. Two known P2X7 antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of hP2X7 receptors.

Identification and functional characterization of cereblon as a binding protein for large-conductance calcium-activated potassium channel in rat brain.

Large-conductance Ca2+-activated K+ (BK(Ca)) channels are activated by membrane depolarization and modulated by intracellular Ca2+. Here, we report the direct interaction of cereblon (CRBN) with the cytosolic carboxy-terminus of the BK(Ca) channel alpha subunit (Slo). Rat CRBN contained the N-terminal domain of the Lon protease, a 'regulators of G protein-signaling' (RGS)-like domain, a leucine zipper (LZ) motif, and four putative protein kinase C (PKC) phosphorylation sites. RNA messages of rat cereblon (rCRBN) were widely distributed in different tissues with especially high-levels of expression in the brain. Direct association of rCRBN with the BK(Ca) channel was confirmed by immunoprecipitation in brain lysate, and the two proteins were co-localized in cultured rat hippocampal neurons. Ionic currents evoked by the rSlo channel were dramatically suppressed upon coexpression of rCRBN. rCRBN decreased the formation of the tetrameric rSlo complex thus reducing the surface expression of functional channels. Therefore, we suggest that CRBN may play an important role in assembly and surface expression of functional BK(Ca) channels by direct interaction with the cytosolic C-terminus of its alpha-subunit.

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase.

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.