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author:("mklann, Eric")
1.  BONLAC: A Combinatorial Proteomic Technique to Measure Stimulus-induced Translational Profiles in Brain Slices 
Neuropharmacology  2015;100:76-89.
Stimulus-triggered protein synthesis is critical for brain health and function. However, due to technical hurdles, de novo neuronal translation is predominantly studied in cultured cells, whereas electrophysiological and circuit analyses often are performed in brain slices. The different properties of these two experimental systems create an information gap about stimulus-induced alterations in the expression of new proteins in mature circuits. To address this, we adapted two existing techniques, BONCAT and SILAC, to a combined proteomic technique, BONLAC, for use in acute adult hippocampal slices. Using BDNF-induced protein synthesis as a proof of concept, we found alterations in expression of proteins involved in neurotransmission, trafficking, and cation binding that differed from those found in a similar screen in cultured neurons. Our results indicate important differences between cultured neurons and slices, and suggest that BONLAC could be used to dissect proteomic changes underlying synaptic events in adult circuits.
PMCID: PMC4584208  PMID: 26205778
BONLAC; BONCAT; SILAC; de novo proteomics; BDNF; hippocampal slice synaptic plasticity; protein synthesis
2.  Translational Control of Long-Lasting Synaptic Plasticity and Memory 
Neuron  2009;61(1):10-26.
Long-lasting forms of synaptic plasticity and memory are dependent on new protein synthesis. Recent advances obtained from genetic, physiological, pharmacological, and biochemical studies provide strong evidence that translational control plays a key role in regulating long-term changes in neural circuits and thus long-term modifications in behavior. Translational control is important for regulating both general protein synthesis and synthesis of specific proteins in response to neuronal activity. In this review, we summarize and discuss recent progress in the field and highlight the prospects for better understanding of long-lasting changes in synaptic strength, learning, and memory and implications for neurological diseases.
PMCID: PMC5154738  PMID: 19146809 CAMSID: cams953
3.  eIF4E/Fmr1 Double Mutant Mice Display Cognitive Impairment in Addition to ASD-like Behaviors 
Neurobiology of disease  2015;83:67-74.
Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse, and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD.
PMCID: PMC4674395  PMID: 26306459
Autism spectrum disorders; protein synthesis; eIF4E; Fragile X syndrome; cognition; genetic models; rodent behavior
4.  Calmodulin activity regulates group I metabotropic glutamate receptor-mediated signal transduction and synaptic depression 
Journal of neuroscience research  2016;94(5):401-408.
Group I metabotropic glutamate receptors (GpI mGluR) including mGluR1 and 5 (mGluR1/5) are coupled to Gq and modulate activity-dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation-dependent long-term depression (LTD). Although it has been established that intracellular Ca2+ and the Gq-regulated signaling molecules are required for mGluR1/5-LTD, whether and how Ca2+ regulates Gq signaling and up-regulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of a Ca2+ sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor W13 (N-[4-Aminobutyl]-5-chloro-2-naphthalenesulfonamide hydrochloride) suppressed the mGluR1/5-stimulated activation of ERK1/2 (extracellular signal-regulated kinase½) and S6K1 (p70-S6 kinase 1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist-induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca2+/CaM-dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Further, disruption of the CaM-CaMK-ERK1/2 signaling cascade suppressed the mGluR1/5-stimulated up-regulation of Arc expression. Together, our data suggest CaM as a new Gq signaling component to couple Ca2+ and protein up-regulation and regulate mGluR1/5-mediated synaptic modification.
Graphical abstract
PMCID: PMC4801747  PMID: 26864654
AB_331647; AB_823494; AB_2265913; AB_823592; AB_887694; Arc; calmodulin; mGluR1/5; synaptic depression; ERK1/2; signal transduction
5.  Mitochondrial Superoxide Contributes to Hippocampal Synaptic Dysfunction and Memory Deficits in Angelman Syndrome Model Mice 
The Journal of Neuroscience  2015;35(49):16213-16220.
Angelman syndrome (AS) is a neurodevelopmental disorder associated with developmental delay, lack of speech, motor dysfunction, and epilepsy. In the majority of the patients, AS is caused by the deletion of small portions of maternal chromosome 15 harboring the UBE3A gene. This results in a lack of expression of the UBE3A gene because the paternal allele is genetically imprinted. The UBE3A gene encodes an enzyme termed ubiquitin ligase E3A (E6-AP) that targets proteins for degradation by the 26S proteasome. Because neurodegenerative disease and other neurodevelopmental disorders have been linked to oxidative stress, we asked whether mitochondrial reactive oxygen species (ROS) played a role in impaired synaptic plasticity and memory deficits exhibited by AS model mice. We discovered that AS mice have increased levels of superoxide in area CA1 of the hippocampus that is reduced by MitoQ 10-methanesuflonate (MitoQ), a mitochondria-specific antioxidant. In addition, we found that MitoQ rescued impairments in hippocampal synaptic plasticity and deficits in contextual fear memory exhibited by AS model mice. Our findings suggest that mitochondria-derived oxidative stress contributes to hippocampal pathophysiology in AS model mice and that targeting mitochondrial ROS pharmacologically could benefit individuals with AS.
SIGNIFICANCE STATEMENT Oxidative stress has been hypothesized to contribute to the pathophysiology of neurodevelopmental disorders, including autism spectrum disorders and Angelman syndrome (AS). Herein, we report that AS model mice exhibit elevated levels of mitochondria-derived reactive oxygen species in pyramidal neurons in hippocampal area CA1. Moreover, we demonstrate that the administration of MitoQ (MitoQ 10-methanesuflonate), a mitochondria-specific antioxidant, to AS model mice normalizes synaptic plasticity and restores memory. Finally, our findings suggest that antioxidants that target the mitochondria could be used therapeutically to ameliorate synaptic and cognitive deficits in individuals with AS.
PMCID: PMC4682786  PMID: 26658871
hippocampus; memory; mitochondria; neurodevelopmental disorders; oxidative stress; synpatic plasticity
6.  Increased expression of the PI3K enhancer PIKE mediates deficits in synaptic plasticity and behavior in Fragile X syndrome 
Cell reports  2015;11(5):727-736.
The PI3K enhancer PIKE links PI3K catalytic subunits to metabotropic glutamate receptors (mGlu) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability Fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity, and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS.
PMCID: PMC4418204  PMID: 25921541
7.  Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism 
The Journal of Neuroscience  2015;35(41):13836-13842.
The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiological and pathological conditions. Although many ASDs arise as a result of mutations in genes with multiple molecular functions, they appear to converge on common biological pathways that give rise to autism-relevant behaviors. Dysregulation of mTOR signaling has been identified as a phenotypic feature common to fragile X syndrome, tuberous sclerosis complex 1 and 2, neurofibromatosis 1, phosphatase and tensin homolog, and potentially Rett syndrome. Below are a summary of topics covered in a symposium that presents dysregulation of mTOR as a unifying theme in a subset of ASDs.
PMCID: PMC4604222  PMID: 26468183
mTOR signaling; Fragile X syndromes; protein synthesis pathway; mouse models of autism
8.  RCAN1 overexpression promotes age-dependent mitochondrial dysregulation related to neurodegeneration in Alzheimer’s disease 
Acta neuropathologica  2015;130(6):829-843.
Aging is the largest risk factor for Alzheimer’s disease (AD). Patients with Down syndrome (DS) develop symptoms consistent with early-onset AD, suggesting that overexpression of chromosome 21 genes such as Regulator of Calcineurin 1 (RCAN1) plays a role in AD pathogenesis. RCAN1 levels are increased in the brain of DS and AD patients but also in the human brain with normal aging. RCAN1 has been implicated in several neuronal functions, but whether its increased expression is correlative or causal in the aging-related progression of AD remains elusive. We show that brain-specific overexpression of the human RCAN1.1S isoform in mice promotes early age-dependent memory and synaptic plasticity deficits, tau pathology, and dysregulation of dynamin-related protein 1 (DRP1) activity associated with mitochondrial dysfunction and oxidative stress, reproducing key AD features. Based on these findings, we propose that chronic RCAN1 overexpression during aging alters DRP1-mediated mitochondrial fission and thus acts to promote AD-related progressive neurodegeneration.
PMCID: PMC4782929  PMID: 26497675
RCAN1; DRP1; Mitochondria; Fission; Calcineurin; Aging
9.  Deconstructing brain-derived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology 
Neural Regeneration Research  2016;11(3):363-367.
Brain-derived neurotrophic factor (BDNF) plays an important role in neurodevelopment, synaptic plasticity, learning and memory, and in preventing neurodegeneration. Despite decades of investigations into downstream signaling cascades and changes in cellular processes, the mechanisms of how BDNF reshapes circuits in vivo remain unclear. This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures, while the majority of studies on synaptic plasticity, learning and memory were performed in acute brain slices or in vivo. A recent study by Bowling-Bhattacharya et al., measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice. In this paper, we place these findings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.
PMCID: PMC4828984  PMID: 27127458
BONLAC; BDNF; adult slice proteomics; neuroproteomics; SILAC; BONCAT; hippocampus; protein synthesis
10.  Dysregulation and restoration of translational homeostasis in fragile X syndrome 
Nature reviews. Neuroscience  2015;16(10):595-605.
Fragile X syndrome (FXS), the most-frequently inherited form of intellectual disability and the most-prevalent single-gene cause of autism, results from a lack of fragile X mental retardation protein (FMRP), an RNA-binding protein that acts, in most cases, to repress translation. Multiple pharmacological and genetic manipulations that target receptors, scaffolding proteins, kinases and translational control proteins can rescue neuronal morphology, synaptic function and behavioural phenotypes in FXS model mice, presumably by reducing excessive neuronal translation to normal levels. Such rescue strategies might also be explored in the future to identify the mRNAs that are critical for FXS pathophysiology.
PMCID: PMC4688896  PMID: 26350240
11.  Identification of Fragile X Syndrome-Specific Molecular Markers in Human Fibroblasts: A Useful Model to Test the Efficacy of Therapeutic Drugs 
Human mutation  2014;35(12):1485-1494.
Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, FMRP, an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout (KO) mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2) and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). Treatment with small molecules that inhibit S6K1, and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.
PMCID: PMC4287266  PMID: 25224527
Fragile X syndrome; FMR1; FMRP; protein synthesis; S6K1; fibroblasts
12.  In-Depth Quantitative Proteomic Analysis of de Novo Protein Synthesis Induced by Brain-Derived Neurotrophic Factor 
Journal of Proteome Research  2014;13(12):5707-5714.
Measuring the synthesis of new proteins in the context of a much greater number of pre-existing proteins can be difficult. To overcome this obstacle, bioorthogonal noncanonical amino acid tagging (BONCAT) can be combined with stable isotope labeling by amino acid in cell culture (SILAC) for comparative proteomic analysis of de novo protein synthesis (BONLAC). In the present study, we show that alkyne resin-based isolation of l-azidohomoalanine (AHA)-labeled proteins using azide/alkyne cycloaddition minimizes contamination from pre-existing proteins. Using this approach, we isolated and identified 7414 BONCAT-labeled proteins. The nascent proteome isolated by BONCAT was very similar to the steady-state proteome, although transcription factors were highly enriched by BONCAT. About 30% of the methionine residues were replaced by AHA in our BONCAT samples, which allowed for identification of methionine-containing peptides. There was no bias against low-methionine proteins by BONCAT at the proteome level. When we applied the BONLAC approach to screen for brain-derived neurotrophic factor (BDNF)-induced protein synthesis, 53 proteins were found to be significantly changed 2 h after BDNF stimulation. Our study demonstrated that the newly synthesized proteome, even after a short period of stimulation, can be efficiently isolated by BONCAT and analyzed to a depth that is similar to that of the steady-state proteome.
PMCID: PMC4261974  PMID: 25271054
BONCAT; pulsed SILAC; mass spectrometry; proteomics; BDNF; translation
13.  Effects of Acute Sleep Deprivation on Motor and Reversal Learning in Mice 
Sleep supports the formation of a variety of declarative and non-declarative memories, and sleep deprivation often impairs these types of memories. In human subjects, natural sleep either during a nap or overnight leads to long-lasting improvements in visuomotor and fine motor tasks, but rodent models recapitulating these findings have been scarce. Here we present evidence that 5 hours of acute sleep deprivation impairs mouse skilled reach learning compared to a matched period of ad libitum sleep. In sleeping mice, the duration of total sleep time during the 5 hours of sleep opportunity or during the first bout of sleep did not correlate with ultimate gain in motor performance. In addition, we observed that reversal learning during the skilled reaching task was also affected by sleep deprivation. Consistent with this observation, 5 hours of sleep deprivation also impaired reversal learning in the water-based Y-maze. In conclusion, acute sleep deprivation negatively impacts subsequent motor and reversal learning and memory.
PMCID: PMC4143485  PMID: 25046627
sleep deprivation; skilled reach learning; reversal learning; Y-maze; motor learning
14.  PERK: a novel therapeutic target for neurodegenerative diseases? 
Identification of therapeutic targets based on novel mechanistic studies is urgently needed for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and prion disease. Multiple lines of evidence have emerged to suggest that inhibition of the stress-induced endoplasmic reticulum kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase) is a potential therapeutic strategy for these diseases. A recently published study demonstrated that oral treatment with a newly characterized PERK inhibitor was able to rescue disease phenotypes displayed in prion disease model mice. Here, we discuss the background and rationale for targeting PERK as a viable therapeutic approach as well as implications of these findings for other neurodegenerative diseases. The promise and caveats of applying this strategy for disease therapy also are discussed.
PMCID: PMC4075240  PMID: 25031640
15.  Prohibitin viral gene transfer protects hippocampal CA1 neurons from ischemia and ameliorates post-ischemic hippocampal dysfunction 
Background and Purpose
Prohibitin (PHB) is a multi-functional protein involved in numerous cellular activities. PHB overexpression protects neurons from injury in vitro, but it is unclear whether PHB can protect selectively vulnerable hippocampal CA1 neurons in a clinically relevant injury model in vivo and, if so, whether the salvaged neurons remain functional.
A mouse model of transient forebrain ischemia that mimics the brain damage produced by cardiac arrest in humans was used to test whether PHB expression protects CA1 neurons from injury. PHB-expressing viral vector was microinjected in mouse hippocampus to upregulate PHB.
PHB overexpression protected CA1 neurons from transient forebrain ischemia. The protection was associated with dampened post-ischemic ROS generation, reduced mitochondrial cytochrome c release and decreased caspase-3 activation. Importantly, the improvement in CA1 neuronal viability translated into an improvement in hippocampal function: PHB expression ameliorated the spatial memory deficit induced by ischemia, assessed by the Y-maze test, and restored post-ischemic synaptic plasticity assessed by long-term potentiation, indicating that the neurons spared form ischemic damage were functionally competent.
These data demonstrate that PHB overexpression protects highly vulnerable CA1 neurons from ischemic injury in vivo, and suggest that the effect is mediated by reduction of post-ischemic ROS generation and preservation of mitochondrial outer membrane integrity that prevents activation of apoptosis. Measures to enhance PHB expression could have translational value in ischemic brain injury and, possibly, other forms of brain injury associated with mitochondrial dysfunction.
PMCID: PMC3971834  PMID: 24619393
prohibitin; Mitochondria; neuroprotection; transient forebrain ischemia
16.  Inhibition of AMP-Activated Protein Kinase Signaling Alleviates Impairments in Hippocampal Synaptic Plasticity Induced by Amyloid β 
The Journal of Neuroscience  2014;34(36):12230-12238.
The AMP-activated protein kinase (AMPK) is a Ser/Thr kinase that is activated in response to low-energy states to coordinate multiple signaling pathways to maintain cellular energy homeostasis. Dysregulation of AMPK signaling has been observed in Alzheimer's disease (AD), which is associated with abnormal neuronal energy metabolism. In the current study we tested the hypothesis that aberrant AMPK signaling underlies AD-associated synaptic plasticity impairments by using pharmacological and genetic approaches. We found that amyloid β (Aβ)-induced inhibition of long-term potentiation (LTP) and enhancement of long-term depression were corrected by the AMPK inhibitor compound C (CC). Similarly, LTP impairments in APP/PS1 transgenic mice that model AD were improved by CC treatment. In addition, Aβ-induced LTP failure was prevented in mice with genetic deletion of the AMPK α2-subunit, the predominant AMPK catalytic subunit in the brain. Furthermore, we found that eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K are key downstream effectors that mediate the detrimental effects of hyperactive AMPK in AD pathophysiology. Our findings describe a previously unrecognized role of aberrant AMPK signaling in AD-related synaptic pathophysiology and reveal a potential therapeutic target for AD.
PMCID: PMC4152616  PMID: 25186765
Alzheimer's disease; long-term potentiation; neurodegeneration; protein synthesis; signaling; translation
17.  Requirement of Mammalian Target of Rapamycin Complex 1 Downstream Effectors in Cued Fear Memory Reconsolidation and Its Persistence 
The Journal of Neuroscience  2014;34(27):9034-9039.
Memory retrieval, often termed reconsolidation, can render previously consolidated memories susceptible to manipulation that can lead to alterations in memory strength. Although it is known that reconsolidation requires mammalian target of rapamycin complex 1 (mTORC1)-dependent translation, the specific contributions of its downstream effectors in reconsolidation are unclear. Using auditory fear conditioning in mice, we investigated the role of eukaryotic translation initiation factor 4E (eIF4E)–eIF4G interactions and p70 S6 kinase polypeptide 1 (S6K1) in reconsolidation. We found that neither 4EGI-1 (2-[(4-(3,4-dichlorophenyl)-thiazol-2-ylhydrazono)-3-(2-nitrophenyl)]propionic acid), an inhibitor of eFI4E–eIF4G interactions, nor PF-4708671 [2-((4-(5-ethylpyrimidin-4-yl)piperazin-1-yl)methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole], an inhibitor of S6K1, alone blocked the reconsolidation of auditory fear memory. In contrast, using these drugs in concert to simultaneously block eIF4E–eIF4G interactions and S6K1 immediately after memory reactivation significantly attenuated fear memory reconsolidation. Moreover, the combination of 4EGI-1 and PF-4708671 further destabilized fear memory 10 d after memory reactivation, which was consistent with experiments using rapamycin, an mTORC1 inhibitor. Furthermore, inhibition of S6K1 immediately after retrieval resulted in memory destabilization 10 d after reactivation, whereas inhibition of eIF4E–eIF4G interactions did not. These results indicate that the reconsolidation of fear memory requires concomitant association of eIF4E to eIF4G as well as S6K1 activity and that the persistence of memory at longer intervals after memory reactivation also requires mTORC1-dependent processes that involve S6K1. These findings suggest a potential mechanism for how mTORC1-dependent translation is fine tuned to alter memory persistence.
PMCID: PMC4078080  PMID: 24990923
consolidation; fear conditioning; long-term memory; mTORC1; reconsolidation; translation
18.  How ketamine helps to overcome depression 
eLife  null;3:e05418.
Genetically modified mice shed new light on how ketamine can target NMDA receptors in the brain to reduce the symptoms of depression.
PMCID: PMC4264404  PMID: 25497432
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat
19.  Translational Control by eIF2α Kinases in Long-lasting Synaptic Plasticity and Long-term Memory 
Although the requirement for new protein synthesis in synaptic plasticity and memory has been well established, recent genetic, molecular, electrophysiological, and pharmacological studies have broadened our understanding of the translational control mechanisms that are involved in these processes. One of the critical translational control points mediating general and gene-specific translation depends on the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) by four regulatory kinases. Here, we review the literature highlighting the important role for proper translational control via regulation of eIF2α phosphorylation by its kinases in long-lasting synaptic plasticity and long-term memory.
PMCID: PMC3769507  PMID: 23707798
protein synthesis; translation initiation; synaptic plasticity; learning; memory; knockout mouse; eIF2; GCN2; PERK; PKR; HRI
20.  Antipsychotics Activate mTORC1-Dependent Translation to Enhance Neuronal Morphological Complexity 
Science signaling  2014;7(308):ra4.
Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. Here, we showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D2R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D2R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics.
PMCID: PMC4063438  PMID: 24425786
21.  Genetic and Acute CPEB Depletion Ameliorate Fragile X Pathophysiology 
Nature medicine  2013;19(11):1473-1477.
Fragile X Syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of Fmr1, which encodes the translational repressor protein FMRP. FMRP and CPEB, an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs, and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1-/y Cpeb−/− double knockout mice displayed significant amelioration of biochemical, morphological, electrophysiological, and behavioral phenotypes associated with FXS. Acute depletion of CPEB in the hippocampus of Fmr1 -/y mice rescued working memory deficits, demonstrating reversal of this FXS phenotype in adults. Finally, we find that FMRP and CPEB balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS, and that the maintenance of this homeostasis by FMRP and CPEB is necessary for normal neurologic function.
PMCID: PMC3823751  PMID: 24141422
22.  The Translation of Translational Control by FMRP: Therapeutic Targets for Fragile X Syndrome 
Nature neuroscience  2013;16(11):1530-1536.
De novo protein synthesis is necessary for long-lasting modifications in synaptic strength and dendritic spine dynamics that underlie cognition1. Fragile X syndrome, characterized by intellectual disability and autistic behaviors, holds promise for revealing the molecular basis for these long-term changes in neuronal function. Loss-of-function of FMRP, the fragile X mental retardation protein, results in defects in synaptic plasticity and cognition in many models of the disease. FMRP is a polyribosome-associated RNA binding protein that regulates the synthesis of a set of plasticity-related proteins by stalling ribosomal translocation on target mRNAs. The recent identification of mRNA targets of FMRP and its upstream regulators, and the use of small molecules to stall ribosomes in the absence of FMRP, have the potential to be translated into novel therapeutic avenues for the treatment of FXS.
PMCID: PMC3999698  PMID: 23584741
23.  Regulator of Calcineurin 1 Modulates Expression of Innate Anxiety and Anxiogenic Responses to Selective Serotonin Reuptake Inhibitor Treatment 
The Journal of Neuroscience  2013;33(43):16930-16944.
Regulator of calcineurin 1 (RCAN1) controls the activity of calcium/calmodulin-dependent phosphatase calcineurin (CaN), which has been implicated in human anxiety disorders. Previously, we reported that RCAN1 functioned as an inhibitor of CaN activity in the brain. However, we now find enhanced phosphorylation of a CaN substrate, cAMP response element-binding protein (CREB), in the brains of Rcan1 knock-out (KO) mice. Consistent with enhanced CREB activation, we also observe enhanced expression of a CREB transcriptional target, brain-derived neurotrophic factor (BDNF) in Rcan1 KO mice. We also discovered that RCAN1 deletion or blockade of RCAN1–CaN interaction reduced CaN and protein phosphatase-1 localization to nuclear-enriched protein fractions and promoted CREB activation. Because of the potential links between CREB, BDNF, and anxiety, we examined the role of RCAN1 in the expression of innate anxiety. Rcan1 KO mice displayed reduced anxiety in several tests of unconditioned anxiety. Acute pharmacological inhibition of CaN rescued these deficits while transgenic overexpression of human RCAN1 increased anxiety. Finally, we found that Rcan1 KO mice lacked the early anxiogenic response to the selective serotonin reuptake inhibitor (SSRI) fluoxetine and had improved latency for its therapeutic anxiolytic effects. Together, our study suggests that RCAN1 plays an important role in the expression of anxiety-related and SSRI-related behaviors through CaN-dependent signaling pathways. These results identify RCAN1 as a mediator of innate emotional states and possible therapeutic target for anxiety.
PMCID: PMC3807023  PMID: 24155299
24.  Suppression of eIF2α kinases alleviates AD-related synaptic plasticity and spatial memory deficits 
Nature neuroscience  2013;16(9):1299-1305.
Expression of long-lasting synaptic plasticity and long-term memory requires new protein synthesis, which can be repressed by phosphorylation of eukaryotic initiation factor 2α subunit (eIF2α). It was reported previously that eIF2α phosphorylation is elevated in the brains of Alzheimer’s disease (AD) patients and AD model mice. Therefore, we determined whether suppressing eIF2α kinases could alleviate synaptic plasticity and memory deficits in AD model mice. The genetic deletion of the eIF2α kinase PERK prevented enhanced eIF2α phosphorylation, as well as deficits in protein synthesis, synaptic plasticity, and spatial memory in APP/PS1 AD model mice. Similarly, deletion of another eIF2α kinase, GCN2, prevented impairments of synaptic plasticity and spatial memory defects displayed in the APP/PS1 mice. Our findings implicate aberrant eIF2α phosphorylation as a novel molecular mechanism underlying AD-related synaptic pathophysioloy and memory dysfunction and suggest that PERK and GCN2 are potential therapeutic targets for the treatment of individuals with AD.
PMCID: PMC3756900  PMID: 23933749
25.  Neuronal Expression of the Ubiquitin E3 ligase APC/C-Cdh1 During Development is Required for Long-term Potentiation, Behavioral Flexibility, and Extinction 
Cdh1 is a regulatory subunit of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin E3 ligase known to be involved in regulating cell cycle progression. Recent studies have demonstrated a role for Cdh1 in neurons during developmental and adult synaptic plasticity, as well as memory. In order to better characterize the contribution of Cdh1 in synaptic plasticity and memory, we generated conditional knockout mice using a neuron-specific enolase (Nse) promoter where Cdh1 was eliminated in neurons from the onset of differentiation. Although we detected impaired long-term potentiation (LTP) in hippocampal slices from the Nse-Cdh1 knockout (KO) mice, performance on several hippocampus-dependent memory tasks remained intact. However, the Nse-Cdh1 KO mice exhibited impaired behavioral flexibility and extinction of previously consolidated memories. These findings suggest a role for Cdh1 in regulating the updating of consolidated memories.
PMCID: PMC3562375  PMID: 23238556

Results 1-25 (66)