SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

Inherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

CC-401 Promotes ß-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.

Pharmacologic expansion of endogenous ß cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control ß-cell growth we screened ∼2400 bioactive compounds for rat ß-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat ß-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) ß-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat ß cells, which were broadly growth responsive to compound treatment, human ß-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3ß (GSK-3ß) or activin A receptor type II-like kinase/transforming growth factor-ß (ALK5/TGF-ß) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human ß-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced ß-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the ß-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control ß-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control ß-cell growth.

Genetic Disruption of Adenosine Kinase in Mouse Pancreatic ß-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance.

Islet ß-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of ß-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate ß-cell replication. Here, we evaluated whether ADK disruption in mouse ß-cells affects ß-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT1Lphi mice to enable constitutive (ßADKO) and conditional (ißADKO) disruption of ADK expression in ß-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, ß-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult ßADKO and ißADKO mice displayed glucose tolerance, insulin tolerance and ß-cell mass comparable to control animals. By contrast, HFD-fed ßADKO and ißADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased ß-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive ß-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive ß-cell response.

A High-content In Vitro Pancreatic Islet ß-cell Replication Discovery Platform.

Loss of insulin-producing ß-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet ß-cells remains an attractive strategy. ß-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain ß-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify ß-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a ß-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on ß-cells and the molecular mechanisms that regulate ß-cell growth.

Repurposing cAMP-modulating medications to promote ß-cell replication.

Loss of ß-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote ß-cell regeneration is a priority. cAMP is an intracellular second messenger that modulates ß-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate ß-cell growth. To identify cAMP-stabilizing medications that promote ß-cell replication, we performed high-content screening of a phosphodiesterase (PDE) inhibitor library. PDE3, -4, and -10 inhibitors, including dipyridamole, were found to promote ß-cell replication in an adenosine receptor-dependent manner. Dipyridamole's action is specific for ß-cells and not α-cells. Next we demonstrated that norepinephrine (NE), a physiologic suppressor of cAMP synthesis in ß-cells, impairs ß-cell replication via activation of α(2)-adrenergic receptors. Accordingly, mirtazapine, an α(2)-adrenergic receptor antagonist and antidepressant, prevents NE-dependent suppression of ß-cell replication. Interestingly, NE's growth-suppressive effect is modulated by endogenously expressed catecholamine-inactivating enzymes (catechol-O-methyltransferase and l-monoamine oxidase) and is dominant over the growth-promoting effects of PDE inhibitors. Treatment with dipyridamole and/or mirtazapine promote ß-cell replication in mice, and treatment with dipyridamole is associated with reduced glucose levels in humans. This work provides new mechanistic insights into cAMP-dependent growth regulation of ß-cells and highlights the potential of commonly prescribed medications to influence ß-cell growth.

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor.

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.

AMPA receptor binding cleft mutations that alter affinity, efficacy, and recovery from desensitization.

Glutamate binds to AMPA receptors within a deep cleft between two globular protein domains (domains 1 and 2). Once glutamate binds, the cleft closes, and agonist-bound structures of the isolated ligand binding core suggest that closure of the binding cleft is sufficiently complete that it essentially prevents ligand dissociation. There is also considerable evidence supporting the view that cleft closure is the initial conformational change that triggers receptor activation and desensitization, and it has been clearly demonstrated that there is a correlation between the degree of cleft closure and agonist efficacy. It is unknown, however, whether the stability of binding cleft closure also influences receptor-channel properties. The crystallographic structures indicate that closed-cleft conformations are stabilized by the formation of hydrogen bonds that involve amino acid side chains of residues in domains 1 and 2. We show here that mutations that disrupt one such cross-cleft hydrogen bond (in the AMPA receptor subunit GluR2) decrease both agonist affinity and efficacy. The same mutations also hasten recovery from desensitization. We conclude that the stability of binding cleft closure has a significant impact on AMPA receptor function and is a major determinant of the apparent affinity of agonists. The results suggest that the stability of cleft closure has been tuned so that glutamate dissociates as rapidly as possible yet remains a full agonist.

Solution X-ray scattering evidence for agonist- and antagonist-induced modulation of cleft closure in a glutamate receptor ligand-binding domain.

Agonist-induced conformational changes in the ligand-binding domains (LBD) of glutamate receptor ion channels provide the driving force for molecular rearrangements that mediate channel opening and subsequent desensitization. The resulting regulated transmembrane ion fluxes form the basis for most excitatory neuronal signaling in the brain. Crystallographic analysis of the GluR2 LBD core has revealed a ligand-binding cleft located between two lobes. Channel antagonists stabilize an open cleft, whereas agonists stabilize a closed cleft. The crystal structure of the apo form is similar to the antagonist-bound, open state. To understand the conformational behavior of the LBD in the absence of crystal lattice constraints, and thus better to appreciate the thermodynamic constraints on ligand binding, we have undertaken a solution x-ray scattering study using two different constructs encoding either the core or an extended LBD. In agreement with the GluR2 crystal structures, the LBD is more compact in the presence of agonist than it is in the presence of antagonist. However, the time-averaged conformation of the ligand-free core in solution is intermediate between the open, antagonist-bound state and the closed, agonist-bound state, suggesting a conformational equilibrium. Addition of peptide moieties that connect the core domain to the other functional domains in each channel subunit appears to constrain the conformational equilibrium in favor of the open state.

Structure and function of glutamate receptor ion channels.

A vast number of proteins are involved in synaptic function. Many have been cloned and their functional role defined with varying degrees of success, but their number and complexity currently defy any molecular understanding of the physiology of synapses. A beacon of success in this medieval era of synaptic biology is an emerging understanding of the mechanisms underlying the activity of the neurotransmitter receptors for glutamate. Largely as a result of structural studies performed in the past three years we now have a mechanistic explanation for the activation of channel gating by agonists and partial agonists; the process of desensitization, and its block by allosteric modulators, is also mostly explained; and the basis of receptor subtype selectivity is emerging with clarity as more and more structures are solved. In the space of months we have gone from cartoons of postulated mechanisms to hard fact. It is anticipated that this level of understanding will emerge for other synaptic proteins in the coming decade.

Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes.

The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical techniques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully closed conformation in the AMPA-bound form. Our results indicate that agonists can induce a range of conformations in the GluR2 ligand-binding core and that domain closure is directly correlated to channel activation. The partially closed, AMPA-bound conformation of the L650T mutant likely captures the structure of an agonist-bound, inactive state of the receptor. Together with previously solved structures, we have determined a mechanism of agonist binding and subsequent conformational rearrangements.

Mechanism of glutamate receptor desensitization.

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.