Biology subjects

Ron, D.

Publications and source records attributed to Ron, D..

29 records · Page 2Linked to original sources

bioRxiv2020

Brain-Specific Inhibition of mTORC1 by a Dual Drug Strategy: A Novel Approach for the Treatment of Alcohol Use Disorder

Alcohol Use Disorder (AUD) is a devastating psychiatric disorder affecting a large portion of the population. Unfortunately, efficacious medications to treat the disease are limited and thus AUD represents an area of unmet medical need. mTORC1 plays a crucial role in neuroadaptations underlying alcohol use. mTORC1 also contributes to alcohol craving, habit, and relapse. Thus, mTORC1 inhibitors are promising therapeutic agents to treat AUD. However, chronic inhibition of mTORC1 in the periphery produces undesirable side effects in humans, which limit their potential clinical use for the treatment of AUD. To overcome these limitations, we utilized a binary drug strategy in which mice were co-administered the mTORC1 inhibitor RapaLink-1 together with a novel small molecule (RapaBlock) to protect mTORC1 activity in the periphery. We show that the dual administration of RapaLink-1 with RapaBlock, abolishes RapaLink-1-dependent mTORC1 inhibition in the liver and blocks adverse side effects detected in humans including body weight loss, glucose intolerance and liver toxicity. Importantly, we show that co-administration of RapaLink-1 and RapaBlock inhibits alcohol-dependent mTORC1 activation in the Nucleus Accumbens and robustly moderates the level of alcohol use. Our data present a novel approach that could be used to treat individuals suffering from AUD.

neuroscience↗

bioRxiv2020

ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

The small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(P). ISRIB and eIF2(P) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(P) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(P). ISRIBs efficacy as an ISR inhibitor in cells also depends on presence of eIF2(P). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(P) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2 during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(P) and ISRIB reciprocally opposed each others binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

biochemistry↗

bioRxiv2020

Cargo receptor-assisted endoplasmic reticulum export of pathogenic α1-antitrypsin polymers

Circulating polymers of alpha1-antitrypsin (1AT) are chemo-attractant for neutrophils and contribute to inflammation in pulmonary, vascular and adipose tissues. Cellular factors affecting the intracellular itinerary of mutant polymerogenic 1AT remain obscure. Here, we report on an unbiased genome-wide CRISPR/Cas9 screen for regulators of trafficking of the polymerogenic 1ATH334D variant. Single guide RNAs targeting genes whose inactivation enhanced accumulation of polymeric 1AT were enriched by iterative construction of CRISPR libraries based on genomic DNA from fixed cells selected for high polymer content by fluorescence-activated cell sorting. This approach bypassed the limitation to conventional enrichment schemes imposed by cell fixation and identified 121 genes involved in polymer retention at false discovery rate < 0.1. From that set of genes, the pathway cargo loading into COPII-coated vesicles was overrepresented with 16 significant genes, including two transmembrane cargo receptors, LMAN1 (ERGIG-53) and SURF4. LMAN1 and SURF4-disrupted cells displayed a secretion defect extended beyond 1AT monomers to polymers, whose low-level secretion was especially dependent on SURF4 and correlated with SURF4-1ATH334D physical interaction and with enhanced co-localisation of polymeric 1ATH334D with the endoplasmic reticulum (ER). These findings suggest that ER cargo receptors co-ordinate intracellular progression of 1AT out of the ER and modulate the accumulation of polymeric 1AT not only by controlling the concentration of precursor monomers but also through a previously-unrecognised role in secretion of the polymers themselves.

cell biology↗

bioRxiv2020

cAMP-Fyn signaling in the dorsomedial striatum direct pathway drives excessive alcohol use

Fyn kinase in the dorsomedial striatum (DMS) of rodents plays a central role in mechanisms underlying excessive alcohol intake. The DMS is comprised of medium spiny neurons (MSNs) that project directly (dMSNs) or indirectly (iMSNs) to the substantia nigra. Here, we examined the cell-type specificity of Fyns actions on alcohol use. First, we knocked down Fyn selectively in DMS dMSNs or iMSNs of mice and measured the level of alcohol consumption. We found that downregulation of Fyn in dMSNs, but not in iMSNs, reduces excessive alcohol but not saccharin intake. D1Rs are coupled to Gs/olf, which activate cAMP signaling. To examine whether Fyns actions are mediated through cAMP signaling, DMS dMSNs were infected with GsDREADD, and the activation of Fyn signaling was measured following CNO treatment. We found that remote stimulation of cAMP signaling in DMS dMSNs activates Fyn and promotes the phosphorylation of the Fyn substrate, GluN2B. In contract, remote activation of GsDREADD in DLS dMSNs did not alter Fyn signaling. We then tested whether activation of GsDREADD in DMS dMSNs or iMSNs alters alcohol intake and observed that CNO-dependent activation of GsDREADD in DMS dMSNs but not iMSNs increases alcohol but not saccharin intake. Finally, we examined the contribution of Fyn to GsDREADD-dependent increase in alcohol intake, and found that systemic administration of the Fyn inhibitor, AZD0503 blocks GsDREADD-dependent increase in alcohol consumption. Our results suggest that the cAMP-Fyn axis in the DMS dMSNs is a molecular transducer of mechanisms underlying the development of excessive alcohol consumption.

neuroscience↗

bioRxiv2019

Differential Correlation of Serum BDNF and microRNA Content in Rats with Rapid or Late Onset of Heavy Alcohol Use

Heavy alcohol use reduces the levels of the brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of rodents through the upregulation of microRNAs targeting BDNF mRNA. In humans, an inverse correlation exists between circulating blood levels of BDNF and the severity of psychiatric disorders including alcohol abuse. Here, we set out to determine whether a history of heavy alcohol use produces comparable alterations in the blood of rats. We used an intermittent access to 20% alcohol using the 2-bottle choice paradigm (IA20%2BC), and measured circulating levels of BDNF protein and microRNAs in the serum of Long-Evans rats before and after 8-weeks of excessive alcohol intake. We observed that the drinking profile of heavy alcohol users is not unified; Whereas 70% of the rats gradually escalate their alcohol intake (Late Onset), 30% of alcohol users exhibit a very Rapid Onset of drinking (Rapid Onset). We found that serum BDNF levels are negatively correlated with alcohol intake in both Rapid Onset and Late Onset rats. In contrast, increased expression of the microRNAs (miRs) targeting BDNF, miR30a-5p, miR-195-5p, miR191-5p and miR206-3p, was detected only in the Rapid Onset rats. Finally, we report that the alcohol-dependent molecular changes are not due to alterations in platelet number. Our data suggest that rats exhibit both Late and Rapid Onset of alcohol intake. We further show that heavy alcohol use produces comparable changes in BDNF protein levels in both groups. However, circulating microRNAs are responsive to alcohol only in the Rapid Onset rats.

neuroscience↗

bioRxiv2019

GDF15 and the beneficial actions of metformin in pre-diabetes

Metformin, the worlds most prescribed anti-diabetic drug, is also effective in preventing Type 2 diabetes in people at high risk, by lowering body weight, fat mass and circulating insulin levels through mechanisms that are incompletely understood. Recent observational studies reporting the association of metformin use and circulating levels of GDF15 led us to hypothesize that GDF15, which signals through a specific receptor complex in the hindbrain to reduce body weight, might mediate these effects. We measured GDF15 in people without diabetes from a randomized placebo-controlled trial of metformin. Over 18 months, participants allocated metformin lost significant weight and levels of GDF15 were persistently elevated compared to placebo. The change in plasma GDF15 in this study correlated positively with weight loss. In wild-type mice, oral metformin increased circulating GDF15 with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to high fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GFRAL. In obese, high fat-fed mice, the effects of metformin to reduce body weight were reversed by a GFRAL antagonist antibody. Metformin had effects on both energy intake and energy expenditure that required GDF15. The insulin sensitising effects of metformin determined by insulin tolerance were abolished in mice lacking GDF15. Metformin significantly reduced fasting glucose and insulin levels in wild type but not in mice lacking GDF15. In summary, metformin increases the circulating levels of GDF15, which appears to be necessary for many of its actions as a metabolic chemopreventive agent.

physiology↗

bioRxiv2019

An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP

AMPylation is an inactivating modification that matches the activity of the major endoplasmic reticulum (ER) chaperone BiP to the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICDs activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface or in residues tracing an inhibitory relay from the dimer interface to the enzymes active site favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically-propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Whereas, a reciprocal signal propagated from the nucleotide binding site, provides a mechanism for coupling the oligomeric-state and enzymatic activity of FICD to the energy status of the ER.\n\nImpact StatementUnique amongst known chaperones, the endoplasmic reticulum (ER)-localized Hsp70, BiP, is subject to transient inactivation under conditions of low ER stress by reversible, covalent modification - AMPylation. The enzyme responsible for this modification, FICD, is in fact a bifunctional enzyme with a single active site capable of both AMPylation and deAMPylation. Here we elucidate, by biochemical, biophysical and structural means, the mechanism by which this enzyme is able to switch enzymatic modality: by regulation of its oligomeric state. The oligomeric state-dependent reciprocal regulation of FICD activity is, in turn, sensitive to the ATP/ADP ratio. This allosteric pathway potentially facilitates the sensing of unfolded protein load in the ER and permits the transduction of this signal into a post-translational buffering of ER chaperone activity.

biochemistry↗

bioRxiv2019

mTORC1 IN THE ORBITOFRONTAL CORTEX PROMOTES HABITUAL ALCOHOL SEEKING

The mammalian target of rapamycin complex 1 (mTORCl) plays an important role in dendritic translation, synaptic plasticity, and learning and memory. We previously showed that heavy alcohol use activates mTORC1 in the orbitofrontal cortex (OFC) of rodents. Here, we set out to determine the consequences of alcohol-dependent mTORC1 activation in the OFC. We found that although inhibition of mTORC1 in the OFC does not alter rat alcohol intake per se, it attenuates alcohol seeking. We then tested whether mTORC1 in the OFC is required for goal-directed or habitual alcohol seeking. To do so, rats were trained self-administer alcohol under a random ratio (RR) or a random interval (RI) schedule of reinforcement, which biases toward goal-directed or habitual responding, respectively, and tested whether mTORC1 inhibition alters lever presses following alcohol devaluation. We found that pharmacological inhibition of mTORC1 or knockdown of the adaptor protein, Raptor, did not affect goal-directed alcohol responding but restored sensitivity to devaluation in RI-trained rats. In contrast, habitual responding for sucrose was unaltered by mTORC1 inhibition. These data suggest that mTORC1 in the OFC drives alcohol habit. We then elucidate the mechanism by which mTORC1 is activated by alcohol, and found that the recruitment of GluN2B during alcohol withdrawal stimulates mTORC1 in OFC cFos-positive neurons. Finally, we show that inhibition of GluN2B in the OFC attenuates both alcohol seeking and habitual responding for alcohol. Together, our data suggest that alcohol withdrawal promotes an NMDAR-dependent activation of mTORC1 which in turn drives habitual alcohol seeking.

neuroscience↗

bioRxiv2017

Choice of PP1 catalytic subunit affects neither the requirement for G-actin nor the insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

The integrated stress response (ISR) is regulated by kinases that phosphorylate the subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2P-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2P dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2P-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2P dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.

biochemistry↗

bioRxiv2017

Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, eIF2B

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the {beta} and {delta} regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

molecular biology↗

bioRxiv2016

TriPer, an optical probe tuned to the endoplasmic reticulum, tracks H2O2 consumption by glutathione

The fate of H2O2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic {beta}-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H2O2 signal and eroded {beta}-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.\n\nSignificance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathiones role ER H2O2 metabolism.\n\nHere we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.\n\nThis study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

cell biology↗