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.

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

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 ß paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased ß-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3ß, 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.

A decrease in eukaryotic elongation factor 2 phosphorylation is required for local translation of sensorin and long-term facilitation in Aplysia.

Mechanistic target of rapamycin complex 1 (mTORC1)-dependent protein synthesis is required for many forms of synaptic plasticity and memory, but the downstream pathways important for synaptic plasticity are poorly understood. Long-term facilitation (LTF) in Aplysia is a form of synaptic plasticity that is closely linked to behavioral memory and an attractive model system for examining the important downstream targets for mTORC1 in regulating synaptic plasticity. Although mTORC1-regulated protein synthesis has been strongly linked to translation initiation, translation elongation is also regulated by mTORC1 and LTF leads to an mTORC1-dependent decrease in eukaryotic elongation factor 2 (eEF2) phosphorylation. The purpose of this study is to test the hypothesis that the decrease in eEF2 phosphorylation is required for mTORC1-dependent translation and plasticity. We show that the LTF-induced decrease in eEF2 phosphorylation is blocked by expression of an eEF2 kinase (eEF2K) modified to be resistant to mTORC1 regulation. We found that expression of this modified kinase blocked LTF. LTF requires local protein synthesis of the neuropeptide sensorin and importantly, local sensorin synthesis can be measured using a dendra fluorescent protein containing the 5' and 3' untranslated regions (UTRs) of sensorin. Using this construct, we show that blocking eEF2 dephosphorylation also blocks the increase in local sensorin synthesis. These results identify decreases in eEF2 phosphorylation as a critical downstream effector of mTOR required for long-term plasticity and identify an important translational target regulated by decreases in eEF2 phosphorylation.

Bidirectional regulation of eEF2 phosphorylation controls synaptic plasticity by decoding neuronal activity patterns.

At the sensory-motor neuron synapse of Aplysia, either spaced or continuous (massed) exposure to serotonin (5-HT) induces a form of intermediate-term facilitation (ITF) that requires new protein synthesis but not gene transcription. However, spaced and massed ITF use distinct molecular mechanisms to maintain increased synaptic strength. Synapses activated by spaced applications of 5-HT generate an ITF that depends on persistent protein kinase A (PKA) activity, whereas an ITF produced by massed 5-HT depends on persistent protein kinase C (PKC) activity. In this study, we demonstrate that eukaryotic elongation factor 2 (eEF2), which catalyzes the GTP-dependent translocation of the ribosome during protein synthesis, acts as a biochemical sensor that is tuned to the pattern of neuronal stimulation. Specifically, we find that massed training leads to a PKC-dependent increase in phosphorylation of eEF2, whereas spaced training results in a PKA-dependent decrease in phosphorylation of eEF2. Importantly, by using either pharmacological or dominant-negative strategies to inhibit eEF2 kinase (eEF2K), we were able to block massed 5-HT-dependent increases in eEF2 phosphorylation and subsequent PKC-dependent ITF. In contrast, pharmacological inhibition of eEF2K during the longer period of time required for spaced training was sufficient to reduce eEF2 phosphorylation and induce ITF. Finally, we find that the massed 5-HT-dependent increase in synaptic strength requires translation elongation, but not translation initiation, whereas the spaced 5-HT-dependent increase in synaptic strength is partially dependent on translation initiation. Thus, bidirectional regulation of eEF2 is critical for decoding distinct activity patterns at synapses by activating distinct modes of translation regulation.

A recollection of mTOR signaling in learning and memory.

Mechanistic target of rapamcyin (mTOR) is a central player in cell growth throughout the organism. However, mTOR takes on an additional, more specialized role in the developed neuron, where it regulates the protein synthesis-dependent, plastic changes underlying learning and memory. mTOR is sequestered in two multiprotein complexes (mTORC1 and mTORC2) that have different substrate specificities, thus allowing for distinct functions at synapses. We will examine how learning activates the mTOR complexes, survey the critical effectors of this pathway in the context of synaptic plasticity, and assess whether mTOR plays an instructive or permissive role in generating molecular memory traces.

Serotonin-induced cleavage of the atypical protein kinase C Apl III in Aplysia.

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.

Compartment-specific, differential regulation of eukaryotic elongation factor 2 and its kinase within Aplysia sensory neurons.

Long-term facilitation (LTF) in Aplysia is a leading model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. LTF requires translational control downstream of target of rapamycin complex 1. Our lab has previously shown that treatment with the facilitating neurotransmitter, 5-hydroxytryptamine (5-HT), causes a target of rapamycin complex 1-mediated decrease in phosphorylation of eukaryotic elongation factor 2 (eEF2) within the neurites of sensory neurons involved in LTF. Here, we characterize the Aplysia orthologue of eEF2 kinase (eEF2K). We show that the Aplysia eEF2K orthologue contains an S6 kinase phosphorylation site and that a serine-to-alanine mutation at this site blocks the ability of 5-HT to decrease eEF2 phosphorylation in neurites. We also show that within the soma, 5-HT has the opposite effect, decreasing eEF2K phosphorylation at the S6 kinase site and, concomitantly, increasing eEF2 phosphorylation. Surprisingly, while eEF2K over-expression inhibits translation of a marker for internal ribosome entry site-dependent translation, it stimulates the translation of a marker for cap-dependent translation. This study demonstrates that eEF2 is differentially regulated in separate compartments and contributes to a growing body of evidence that inhibition of elongation can stimulate the translation of certain transcripts.