mGluR5 Negative Modulators for Fragile X: Treatment Resistance and Persistence

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance (“tolerance”) after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

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mGluR5 Negative Modulators for Fragile X: Resistance and Persistence

10.1101/2021.07.02.450894 ◽

2021 ◽

Author(s):

David C. Stoppel ◽

Patrick K. McCamphill ◽

Rebecca K. Senter ◽

Arnold J. Heynen ◽

Mark F. Bear

Keyword(s):

Clinical Trials ◽

Protein Synthesis ◽

Chronic Treatment ◽

Metabotropic Glutamate Receptor ◽

Glycogen Synthase ◽

Fragile X ◽

Treatment Resistance ◽

Chronic Administration ◽

Inhibitory Avoidance ◽

Cross Tolerance

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance (tolerance) after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

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Selective inhibition of glycogen synthase kinase 3α corrects pathophysiology in a mouse model of fragile X syndrome

Science Translational Medicine ◽

10.1126/scitranslmed.aam8572 ◽

2020 ◽

Vol 12 (544) ◽

pp. eaam8572 ◽

Cited By ~ 4

Author(s):

Patrick K. McCamphill ◽

Laura J. Stoppel ◽

Rebecca K. Senter ◽

Michael C. Lewis ◽

Arnold J. Heynen ◽

...

Keyword(s):

Protein Synthesis ◽

Mouse Model ◽

Fragile X Syndrome ◽

Animal Studies ◽

Metabotropic Glutamate Receptor ◽

Glycogen Synthase ◽

Fragile X ◽

Selective Inhibition ◽

Audiogenic Seizures ◽

Glycogen Synthase Kinase

Fragile X syndrome is caused by FMR1 gene silencing and loss of the encoded fragile X mental retardation protein (FMRP), which binds to mRNA and regulates translation. Studies in the Fmr1−/y mouse model of fragile X syndrome indicate that aberrant cerebral protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5) signaling contributes to disease pathogenesis, but clinical trials using mGluR5 inhibitors were not successful. Animal studies suggested that treatment with lithium might be an alternative approach. Targets of lithium include paralogs of glycogen synthase kinase 3 (GSK3), and nonselective small-molecule inhibitors of these enzymes improved disease phenotypes in a fragile X syndrome mouse model. However, the potential therapeutic use of GSK3 inhibitors has been hampered by toxicity arising from inhibition of both α and β paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased β-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3β, corrected aberrant protein synthesis, audiogenic seizures, and sensory cortex hyperexcitability in Fmr1−/y mice. Although inhibiting either paralog prevented induction of NMDA receptor–dependent long-term depression (LTD) in the hippocampus, only inhibition of GSK3α impaired mGluR5-dependent and protein synthesis–dependent LTD. Inhibition of GSK3α additionally corrected deficits in learning and memory in Fmr1−/y mice; unlike mGluR5 inhibitors, there was no evidence of tachyphylaxis or enhanced psychotomimetic-induced hyperlocomotion. GSK3α selective inhibitors may have potential as a therapeutic approach for treating fragile X syndrome.

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Selective role of the translin/trax RNase complex in hippocampal synaptic plasticity

Molecular Brain ◽

10.1186/s13041-020-00691-5 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Alan Jung Park ◽

Mahesh Shivarama Shetty ◽

Jay M. Baraban ◽

Ted Abel

Keyword(s):

Protein Synthesis ◽

Synaptic Plasticity ◽

Molecular Mechanisms ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Local Protein Synthesis ◽

Hippocampal Synaptic Plasticity ◽

Behavioral Abnormalities ◽

Local Protein

Abstract Activity-dependent local protein synthesis is critical for synapse-specific, persistent plasticity. Abnormalities in local protein synthesis have been implicated in psychiatric disorders. We have recently identified the translin/trax microRNA-degrading enzyme as a novel mediator of protein synthesis at activated synapses. Additionally, translin knockout (KO) mice, which lack translin/trax, exhibit some of the behavioral abnormalities found in a mouse model of fragile X syndrome (fragile X mental retardation protein-FMRP-KO mice). Therefore, identifying signaling pathways interacting with translin/trax to support persistent synaptic plasticity is a translationally relevant goal. Here, as a first step to achieve this goal, we have assessed the requirement of translin/trax for multiple hippocampal synaptic plasticity paradigms that rely on distinct molecular mechanisms. We found that mice lacking translin/trax exhibited selective impairment in a form of persistent hippocampal plasticity, which requires postsynaptic protein kinase A (PKA) activity. In contrast, enduring forms of plasticity that are dependent on presynaptic PKA were unaffected. Furthermore, these mice did not display exaggerated metabotropic glutamate receptor-mediated long-term synaptic depression (mGluR-LTD), a hallmark of the FMRP KO mice. On the contrary, translin KO mice exhibited deficits in N-methyl-d-aspartate receptor (NMDAR) dependent LTD, a phenotype not observed in the FMRP knockouts. Taken together, these findings demonstrate that translin/trax mediates long-term synaptic plasticity that is dependent on postsynaptic PKA signaling and suggest that translin/trax and FMRP play distinct roles in hippocampal synaptic plasticity.

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β-Arrestin2 Couples Metabotropic Glutamate Receptor 5 to Neuronal Protein Synthesis and Is a Potential Target to Treat Fragile X

Cell Reports ◽

10.1016/j.celrep.2017.02.075 ◽

2017 ◽

Vol 18 (12) ◽

pp. 2807-2814 ◽

Cited By ~ 24

Author(s):

Laura J. Stoppel ◽

Benjamin D. Auerbach ◽

Rebecca K. Senter ◽

Anthony R. Preza ◽

Robert J. Lefkowitz ◽

...

Keyword(s):

Protein Synthesis ◽

Glutamate Receptor ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Metabotropic Glutamate Receptor 5 ◽

Metabotropic Glutamate ◽

Neuronal Protein ◽

Potential Target

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Selective role of the translin/trax RNase complex in hippocampal synaptic plasticity

10.21203/rs.3.rs-29690/v2 ◽

2020 ◽

Author(s):

Alan Jung Park ◽

Mahesh Shivarama Shetty ◽

Jay M. Baraban ◽

Ted Abel

Keyword(s):

Protein Synthesis ◽

Synaptic Plasticity ◽

Molecular Mechanisms ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Local Protein Synthesis ◽

Hippocampal Synaptic Plasticity ◽

Behavioral Abnormalities ◽

Local Protein

Abstract Activity-dependent local protein synthesis is critical for synapse-specific, persistent plasticity. Abnormalities in local protein synthesis have been implicated in psychiatric disorders. We have recently identified the translin/trax microRNA-degrading enzyme as a novel mediator of protein synthesis at activated synapses. Additionally, translin knockout (KO) mice, which lack translin/trax, exhibit some of the behavioral abnormalities found in a mouse model of fragile X syndrome (fragile X mental retardation protein-FMRP-KO mice). Therefore, identifying signaling pathways interacting with translin/trax to support persistent synaptic plasticity is a translationally relevant goal. Here, as a first step to achieve this goal, we have assessed the requirement of translin/trax for multiple hippocampal synaptic plasticity paradigms that rely on distinct molecular mechanisms. We found that mice lacking translin/trax exhibited selective impairment in a form of persistent hippocampal plasticity, which requires postsynaptic protein kinase A (PKA) activity. In contrast, enduring forms of plasticity that are dependent on presynaptic PKA were unaffected. Furthermore, these mice did not display exaggerated metabotropic glutamate receptor-mediated long-term synaptic depression (mGluR-LTD), a hallmark of the FMRP KO mice. . On the contrary, translin KO mice exhibited deficits in N-methyl-D-aspartate receptor (NMDAR) dependent LTD, a phenotype not observed in the FMRP knockouts. Taken together, these findings demonstrate that translin/trax mediates long-term synaptic plasticity that is dependent on postsynaptic PKA signaling and suggest that translin/trax and FMRP play distinct roles in hippocampal synaptic plasticity.

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Selective role of the translin/trax RNase complex in hippocampal synaptic plasticity

10.21203/rs.3.rs-29690/v1 ◽

2020 ◽

Author(s):

Alan Jung Park ◽

Mahesh Shivarama Shetty ◽

Jay M. Baraban ◽

Ted Abel

Keyword(s):

Protein Synthesis ◽

Synaptic Plasticity ◽

Mouse Model ◽

Fragile X Syndrome ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Local Protein Synthesis ◽

Hippocampal Synaptic Plasticity ◽

Local Protein

Abstract Activity-dependent local protein synthesis is critical for synapse-specific, persistent plasticity. Abnormalities in local protein synthesis have been implicated in psychiatric disorders. We have recently identified the translin/trax microRNA-degrading enzyme as a novel mediator of protein synthesis at activated synapses. Additionally, mice lacking translin/trax exhibit some of the behavioral abnormalities found in a mouse model of fragile X syndrome. Therefore, identifying signaling pathways interacting with translin/trax to support persistent synaptic plasticity is a translationally relevant goal. Here, as a first step to achieve this goal, we have assessed the requirement of translin/trax for multiple hippocampal synaptic plasticity paradigms that rely on distinct molecular mechanisms. We found that mice lacking translin/trax exhibited selective impairment in a form of persistent hippocampal plasticity, which requires postsynaptic PKA activity. In contrast, enduring forms of plasticity that are dependent on presynaptic PKA were unaffected. Furthermore, these mice did not display exaggerated metabotropic glutamate receptor-mediated long-term synaptic depression, a hallmark of the mouse model of fragile X syndrome. Taken together, these findings demonstrate that translin/trax mediates long-term synaptic plasticity that is dependent on postsynaptic PKA signaling.

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Loss of the Fragile X Mental Retardation Protein Decouples Metabotropic Glutamate Receptor Dependent Priming of Long-Term Potentiation From Protein Synthesis

Journal of Neurophysiology ◽

10.1152/jn.00449.2010 ◽

2010 ◽

Vol 104 (2) ◽

pp. 1047-1051 ◽

Cited By ~ 42

Author(s):

Benjamin D. Auerbach ◽

Mark F. Bear

Keyword(s):

Protein Synthesis ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Long Term Potentiation ◽

Metabotropic Glutamate ◽

Fragile X Mental Retardation ◽

Term Potentiation ◽

Mental Retardation Protein ◽

Dependent Protein

Fragile X Syndrome (FXS), the most common inherited form of intellectual disability, is caused by loss of the fragile X mental retardation protein (FMRP). FMRP is a negative regulator of local mRNA translation downstream of group 1 metabotropic glutamate receptor (Gp1 mGluR) activation. In the absence of FMRP there is excessive mGluR-dependent protein synthesis, resulting in exaggerated mGluR-dependent long-term synaptic depression (LTD) in area CA1 of the hippocampus. Understanding disease pathophysiology is critical for development of therapies for FXS and the question arises of whether it is more appropriate to target excessive LTD or excessive mGluR-dependent protein synthesis. Priming of long-term potentiation (LTP) is a qualitatively different functional consequence of Gp1 mGluR-stimulated protein synthesis at the same population of CA1 synapses where LTD can be induced. Therefore we determined if LTP priming, like LTD, is also disrupted in the Fmr1 knockout (KO) mouse. We found that mGluR-dependent priming of LTP is of comparable magnitude in wild-type (WT) and Fmr1 KO mice. However, whereas LTP priming requires acute stimulation of protein synthesis in WT mice, it is no longer protein synthesis dependent in the Fmr1 KO. These experiments show that the dysregulation of mGluR-mediated protein synthesis seen in Fmr1 KO mice has multiple consequences on synaptic plasticity, even within the same population of synapses. Furthermore, it suggests that there is a bifurcation in the Gp1 mGluR signaling pathway, with one arm triggering synaptic modifications such as LTP priming and LTD and the other stimulating protein synthesis that is permissive for these modifications.

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Effect of p-chlorophenylalanine on the acquisition of tolerance to the hypnotic effects of pentobarbital, barbital, and ethanol

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y80-156 ◽

1980 ◽

Vol 58 (9) ◽

pp. 1031-1041 ◽

Cited By ~ 17

Author(s):

J. M. Khanna ◽

H. Kalant ◽

A. D. Lê ◽

J. Mayer ◽

A. E. LeBlanc

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Chronic Treatment ◽

Chronic Administration ◽

Blood Levels ◽

Sodium Pentobarbital ◽

Cross Tolerance ◽

Tolerance Development ◽

Hypnotic Effect ◽

Central Depressant

Rats were given pentobarbital by daily intubation. Sleeping times and blood levels of drug at awakening, after intraperitoneal test doses of ethanol, pentobarbital, or barbital, were measured at various times during chronic treatment in order to assess the degree of tolerance developed. No central nervous system (CNS) tolerance to pentobarbital or cross-tolerance to barbital or ethanol occurred on treatment with sodium pentobarbital, 50 mg/kg, daily. However, when the size and frequency of pentobarbital treatment doses were increased (50–80 mg/kg, three times daily) a clear CNS tolerance to barbital occurred. Chronic administration of p-chlorophenylalanine (p-CPA), in a dose previously shown to maintain more than 95% depletion of brain serotonin (5-HT), enhanced the acute hypnotic effect of barbiturates and ethanol. Independently of this effect, p-CPA treatment also resulted in a reduction in the development of CNS tolerance. These results are consistent with earlier findings that brain 5-HT depletion retards tolerance development to central depressant drugs as measured by a variety of unrelated tests.

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Selective role of the translin/trax RNase complex in hippocampal synaptic plasticity

10.1101/2020.05.19.105189 ◽

2020 ◽

Cited By ~ 1

Author(s):

Alan Jung Park ◽

Mahesh Shivarama Shetty ◽

Jay M. Baraban ◽

Ted Abel

Keyword(s):

Protein Synthesis ◽

Synaptic Plasticity ◽

Mouse Model ◽

Fragile X Syndrome ◽

Metabotropic Glutamate Receptor ◽

Fragile X ◽

Local Protein Synthesis ◽

Hippocampal Synaptic Plasticity ◽

Local Protein

AbstractActivity-dependent local protein synthesis is critical for synapse-specific, persistent plasticity. Abnormalities in local protein synthesis have been implicated in psychiatric disorders. We have recently identified the translin/trax microRNA-degrading enzyme as a novel mediator of protein synthesis at activated synapses. Additionally, mice lacking translin/trax exhibit some of the behavioral abnormalities found in a mouse model of fragile X syndrome. Therefore, identifying signaling pathways interacting with translin/trax to support persistent synaptic plasticity is a translationally relevant goal. Here, as a first step to achieve this goal, we have assessed the requirement of translin/trax for multiple hippocampal synaptic plasticity paradigms that rely on distinct molecular mechanisms. We found that mice lacking translin/trax exhibited selective impairment in a form of persistent hippocampal plasticity, which requires postsynaptic PKA activity. In contrast, enduring forms of plasticity that are dependent on presynaptic PKA were unaffected. Furthermore, these mice did not display exaggerated metabotropic glutamate receptor-mediated long-term synaptic depression, a hallmark of the mouse model of fragile X syndrome. Taken together, these findings demonstrate that translin/trax mediates long-term synaptic plasticity that is dependent on postsynaptic PKA signaling.

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Testing Fmr1KO Phenotypes in Response to GSK3 Inhibitors: SB216763 versus AFC03127

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.751307 ◽

2021 ◽

Vol 14 ◽

Author(s):

Pamela R. Westmark ◽

Beatrice Garrone ◽

Rosella Ombrato ◽

Claudio Milanese ◽

Francesco Paolo Di Giorgio ◽

...

Keyword(s):

Metabotropic Glutamate Receptor ◽

Glycogen Synthase ◽

Fragile X ◽

Drug Dose ◽

Amyloid Beta Protein ◽

Spine Density ◽

Spine Length ◽

Gsk3 Inhibitor

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer’s disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.

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