Transcriptome-wide gene-gene interaction associations elucidate pathways and functional enrichment of complex traits.

It remains unknown to what extent gene-gene interactions contribute to complex traits. Here, we introduce a new approach using predicted gene expression to perform exhaustive transcriptome-wide interaction studies (TWISs) for multiple traits across all pairs of genes expressed in several tissue types. Using imputed transcriptomes, we simultaneously reduce the computational challenge and improve interpretability and statistical power. We discover (in the UK Biobank) and replicate (in independent cohorts) several interaction associations, and find several hub genes with numerous interactions. We also demonstrate that TWIS can identify novel associated genes because genes with many or strong interactions have smaller single-locus model effect sizes. Finally, we develop a method to test gene set enrichment of TWIS associations (E-TWIS), finding numerous pathways and networks enriched in interaction associations. Epistasis is may be widespread, and our procedure represents a tractable framework for beginning to explore gene interactions and identify novel genomic targets.

TDP-43 knockdown in mouse model of ALS leads to dsRNA deposition, gliosis, and neurodegeneration in the spinal cord.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

Novel characterization of the multivariate genetic architecture of internalizing psychopathology and alcohol use.

Genetic correlations suggest that the genetic relationship of alcohol use with internalizing psychopathology depends on the measure of alcohol use. Problematic alcohol use (PAU) is positively genetically correlated with internalizing psychopathology, whereas alcohol consumption ranges from not significantly correlated to moderately negatively correlated with internalizing psychopathology. To explore these different genetic relationships of internalizing psychopathology with alcohol use, we performed a multivariate genome-wide association study of four correlated factors (internalizing psychopathology, PAU, quantity of alcohol consumption, and frequency of alcohol consumption) and then assessed genome-wide and local genetic covariance between these factors. We identified 14 significant regions of local, largely positive, genetic covariance between PAU and internalizing psychopathology and 12 regions of significant local genetic covariance (including both positive and negative genetic covariance) between consumption factors and internalizing psychopathology. Partitioned genetic covariance among functional annotations suggested that brain tissues contribute significantly to positive genetic covariance between internalizing psychopathology and PAU but not to the genetic covariance between internalizing psychopathology and quantity or frequency of alcohol consumption. We hypothesize that genome-wide genetic correlations between alcohol use and psychiatric traits may not capture the more complex shared or divergent genetic architectures at the locus or tissue specific level. This study highlights the complexity of genetic architectures of alcohol use and internalizing psychopathology, and the differing shared genetics of internalizing disorders with PAU compared to consumption.

The Role of A Priori-Identified Addiction and Smoking Gene Sets in Smoking Behaviors.

INTRODUCTION: Smoking is a leading cause of death, and genetic variation contributes to smoking behaviors. Identifying genes and sets of genes that contribute to risk for addiction is necessary to prioritize targets for functional characterization and for personalized medicine. METHODS: We performed a gene set-based association and heritable enrichment study of two addiction-related gene sets, those on the Smokescreen Genotyping Array and the nicotinic acetylcholine receptors, using the largest available GWAS summary statistics. We assessed smoking initiation, cigarettes per day, smoking cessation, and age of smoking initiation. RESULTS: Individual genes within each gene set were significantly associated with smoking behaviors. Both sets of genes were significantly associated with cigarettes per day, smoking initiation, and smoking cessation. Age of initiation was only associated with the Smokescreen gene set. Although both sets of genes were enriched for trait heritability, each accounts for only a small proportion of the single nucleotide polymorphism-based heritability (2%-12%). CONCLUSIONS: These two gene sets are associated with smoking behaviors, but collectively account for a limited amount of the genetic and phenotypic variation of these complex traits, consistent with high polygenicity. IMPLICATIONS: We evaluated evidence for the association and heritable contribution of expert-curated and bioinformatically identified sets of genes related to smoking. Although they impact smoking behaviors, these specifically targeted genes do not account for much of the heritability in smoking and will be of limited use for predictive purposes. Advanced genome-wide approaches and integration of other 'omics data will be needed to fully account for the genetic variation in smoking phenotypes.

Accumulation of Polyribosomes in Dendritic Spine Heads, But Not Bases and Necks, during Memory Consolidation Depends on Cap-Dependent Translation Initiation.

Translation in dendrites is believed to support synaptic changes during memory consolidation. Although translational control mechanisms are fundamental mediators of memory, little is known about their role in local translation. We previously found that polyribosomes accumulate in dendritic spines of the adult rat lateral amygdala (LA) during consolidation of aversive pavlovian conditioning and that this memory requires cap-dependent initiation, a primary point of translational control in eukaryotic cells. Here we used serial electron microscopy reconstructions to quantify polyribosomes in LA dendrites when consolidation was blocked by the cap-dependent initiation inhibitor 4EGI-1. We found that 4EGI-1 depleted polyribosomes in dendritic shafts and selectively prevented their upregulation in spine heads, but not bases and necks, during consolidation. Cap-independent upregulation was specific to spines with small, astrocyte-associated synapses. Our results reveal that cap-dependent initiation is involved in local translation during learning and that local translational control varies with synapse type.SIGNIFICANCE STATEMENT Translation initiation is a central regulator of long-term memory formation. Local translation in dendrites supports memory by providing necessary proteins at synaptic sites, but it is unknown whether this requires initiation or bypasses it. We used serial electron microscopy reconstructions to examine polyribosomes in dendrites when memory formation was blocked by an inhibitor of translation initiation. This revealed two major pools of polyribosomes that were upregulated during memory formation: one pool in dendritic spine heads that was initiation dependent and another pool in the bases and necks of small spines that was initiation independent. Thus, translation regulation differs between spine types and locations, and translation that occurs closest to individual synapses during memory formation is initiation dependent.

RCAN1 overexpression promotes age-dependent mitochondrial dysregulation related to neurodegeneration in Alzheimer's disease.

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.

The eIF2α kinase PERK limits the expression of hippocampal metabotropic glutamate receptor-dependent long-term depression.

The proper regulation of translation is required for the expression of long-lasting synaptic plasticity. A major site of translational control involves the phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by PKR-like endoplasmic reticulum (ER) kinase (PERK). To determine the role of PERK in hippocampal synaptic plasticity, we used the Cre-lox expression system to selectively disrupt PERK expression in the adult mouse forebrain. Here, we demonstrate that in hippocampal area CA1, metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) is associated with increased eIF2α phosphorylation, whereas stimulation of early- and late-phase long-term potentiation (E-LTP and L-LTP, respectively) is associated with decreased eIF2α phosphorylation. Interesting, although PERK-deficient mice exhibit exaggerated mGluR-LTD, both E-LTP and L-LTP remained intact. We also found that mGluR-LTD is associated with a PERK-dependent increase in eIF2α phosphorylation. Our findings are consistent with the notion that eIF2α phosphorylation is a key site for the bidirectional control of persistent forms of synaptic LTP and LTD and suggest a distinct role for PERK in mGluR-LTD.

Regulator of calcineurin 1 modulates expression of innate anxiety and anxiogenic responses to selective serotonin reuptake inhibitor treatment.

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.

cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response.

Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation.

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ~0.9 in as little as six-weeks with a mean firing rate of ~13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of sporadic Alzheimer's disease by mimicking blood-brain barrier breakdown using a human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

AKT2 modulates astrocytic nicotine responses in vivo.

A better understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate the detrimental effects of nicotine withdrawal, and increase successful cessation of use. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4ß2 and α7. We recently found that Protein kinase B-ß (Pkb-ß or Akt2) expression is restricted to astrocytes in mice and humans. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice for in vivo and in vitro experiments. For in vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 µg/mL) and following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. We validated our approaches using lipopolysaccharide (LPS) exposure inducing astrogliosis. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed increased astrocyte morphology complexity. In culture, we found that 100µM nicotine was sufficient for morphological changes and blocking α7 or α4ß2 nAChRs prevented observed morphologic changes. Finally, we performed conditioned place preference (CPP) in Akt2 cKO mice and found that astrocytic AKT2 deficiency reduced nicotine preference compared to controls. These findings show the importance of nAChRs and Akt2 signaling in the astrocytic response to nicotine.

Multiple components of eIF4F are required for protein synthesis-dependent hippocampal long-term potentiation.

Persistent forms of synaptic plasticity are widely thought to require the synthesis of new proteins. This feature of long-lasting forms of plasticity largely has been demonstrated using inhibitors of general protein synthesis, such as either anisomycin or emetine. However, these drugs, which inhibit elongation, cannot address detailed questions about the regulation of translation initiation, where the majority of translational control occurs. Moreover, general protein synthesis inhibitors cannot distinguish between cap-dependent and cap-independent modes of translation initiation. In the present study, we took advantage of two novel compounds, 4EGI-1 and hippuristanol, each of which targets a different component of the eukaryotic initiation factor (eIF)4F initiation complex, and investigated their effects on long-term potentiation (LTP) at CA3-CA1 synapses in the hippocampus. We found that 4EGI-1 and hippuristanol both attenuated long-lasting late-phase LTP induced by two different stimulation paradigms. We also found that 4EGI-1 and hippuristanol each were capable of blocking the expression of newly synthesized proteins immediately after the induction of late-phase LTP. These new pharmacological tools allow for a more precise dissection of the role played by translational control pathways in synaptic plasticity and demonstrate the importance of multiple aspects of eIF4F in processes underlying hippocampal LTP, laying the foundation for future studies investigating the role of eIF4F in hippocampus-dependent memory processes.

Spatial memory deficits and motor coordination facilitation in cGMP-dependent protein kinase type II-deficient mice.

Activity-dependent trafficking of AMPA receptors to synapses regulates synaptic strength. Activation of the NMDA receptor induces several second messenger pathways that contribute to receptor trafficking-dependent plasticity, including the NO pathway, which elevates cGMP. In turn, cGMP activates the cGMP-dependent protein kinase type II (cGKII), which phosphorylates the AMPA receptor subunit GluA1 at serine 845, a critical step facilitating synaptic delivery in the mechanism of activity-dependent synaptic potentiation. Since cGKII is expressed in the striatum, amygdala, cerebral cortex, and hippocampus, it has been proposed that mice lacking cGKII may present phenotypic differences compared to their wild-type littermates in emotion-dependent tasks, learning and memory, and drug reward salience. Previous studies have shown that cGKII KO mice ingest higher amounts of ethanol as well as exhibit elevated anxiety levels compared to wild-type (WT) littermates. Here, we show that cGKII KO mice are significantly deficient in spatial learning while exhibiting facilitated motor coordination, demonstrating a clear dependence of memory-based tasks on cGKII. We also show diminished GluA1 phosphorylation in the postsynaptic density (PSD) of cGKII KO prefrontal cortex while in hippocampal PSD fractions, phosphorylation was not significantly altered. These data suggest that the role of cGKII may be more robust in particular brain regions, thereby impacting complex behaviors dependent on these regions differently.

Characterization and reversal of synaptic defects in the amygdala in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS), a common inherited form of mental impairment and autism, is caused by transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. Earlier studies have identified a role for aberrant synaptic plasticity mediated by the metabotropic glutamate receptors (mGluRs) in FXS. However, many of these observations are derived primarily from studies in the hippocampus. The strong emotional symptoms of FXS, on the other hand, are likely to involve the amygdala. Unfortunately, little is known about how exactly FXS affects synaptic function in the amygdala. Here, using whole-cell recordings in brain slices from adult Fmr1 knockout mice, we find mGluR-dependent long-term potentiation to be impaired at thalamic inputs to principal neurons in the lateral amygdala. Consistent with this long-term potentiation deficit, surface expression of the AMPA receptor subunit, GluR1, is reduced in the lateral amygdala of knockout mice. In addition to these postsynaptic deficits, lower presynaptic release was manifested by a decrease in the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), increased paired-pulse ratio, and slower use-dependent block of NMDA receptor currents. Strikingly, pharmacological inactivation of mGluR5 with 2-methyl-6-phenylethynyl-pyridine (MPEP) fails to rescue either the deficit in long-term potentiation or surface GluR1. However, the same acute MPEP treatment reverses the decrease in mEPSC frequency, a finding of potential therapeutic relevance. Therefore, our results suggest that synaptic defects in the amygdala of knockout mice are still amenable to pharmacological interventions against mGluR5, albeit in a manner not envisioned in the original hippocampal framework.

Diazepam effects on anxiety-related defensive behavior of male and female high and low open-field activity inbred mouse strains.

Open-field activity is a commonly used measure of anxiety-related behavior in rodents. The inbred High and Low Activity strains of mice, selected for extreme differences in open-field activity, have been used as a genetic model of anxiety-related behaviors. These selected strains have been thoroughly studied through extensive behavioral testing, quantitative trait locus (QTL) mapping, whole-genome sequencing, and RNA sequencing, to uncover phenotypic and genotypic differences related to anxiety-related behavior. However, the effects of anxiolytic drugs on anxiety-related behavior in these strains have not been studied previously. This study allowed us to expand on previous findings to further characterize the anxiety-related behavior of these unique strains, using an anxiolytic drug. The goal of this study was to determine whether the treatment of adult male and female High Activity (low anxiety) and Low Activity (high anxiety) mice with diazepam, an agonist at the benzodiazepine allosteric site on the GABAA receptor and a drug commonly prescribed to treat anxiety disorders in humans, led to decreases in anxiety-like defensive behavioral responses as assessed in the open-field test (OFT) and elevated plus-maze (EPM). We tested the effects of three doses of diazepam (0, 0.5, 1.0, 3.0 mg/kg, i.p.), given 30 min before behavioral testing to one High Activity strain (H2) and two Low Activity strains (L1 and L2). There was an anxiolytic effect of diazepam observed in the High Activity strain, with more entries into the open arms of the elevated plus-maze, an effect similar to that seen in common mouse strains. However, the only anxiolytic effect of diazepam seen in the Low Activity strains was a reduction in stretch attend posture (SAP). Low Activity strains also displayed freezing behavior in both the OFT and EPM. The combination of the observed freezing behavior, that was not reduced by diazepam, and the reduction in SAP seen with diazepam, suggests a more complex phenotype that includes a component of innate fear in addition to anxiety-related risk assessment behaviors. Since fear and anxiety are distinguishable traits, and both contribute to human anxiety disorders, these results provide novel insight about interpretation of previous genetic and phenotypic differences observed between the High and Low Activity strains.

Testing associations between human anxiety and genes previously implicated by mouse anxiety models.

Anxiety disorders are common and can be debilitating, with effective treatments remaining hampered by an incomplete understanding of the underlying genetic etiology. Improvements have been made in understanding the genetic influences on mouse behavioral models of anxiety, yet it is unclear the extent to which genes identified in these experimental systems contribute to genetic variation in human anxiety phenotypes. Leveraging new and existing large-scale human genome-wide association studies, we tested whether sets of genes previously identified in mouse anxiety-like behavior studies contribute to a range of human anxiety disorders. When tested as individual genes, 13 mouse-identified genes were associated with human anxiety phenotypes, suggesting an overlap of individual genes contributing to both mouse models of anxiety-like behaviors and human anxiety traits. When genes were tested as sets, we did identify 14 significant associations between mouse gene sets and human anxiety, but the majority of gene sets showed no significant association with human anxiety phenotypes. These few significant associations indicate a need to identify and develop more translatable mouse models by identifying sets of genes that "match" between model systems and specific human phenotypes of interest. We suggest that continuing to develop improved behavioral paradigms and finer-scale experimental data, for instance from individual neuronal subtypes or cell-type-specific expression data, is likely to improve our understanding of the genetic etiology and underlying functional changes in anxiety disorders.

Amyloid ß-induced impairments in hippocampal synaptic plasticity are rescued by decreasing mitochondrial superoxide.

Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer's disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid ß peptide (Aß1-42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by diphenyliodonium (DPI) or by genetically deleting gp91(phox), the key enzymatic component of NADPH oxidase, had no effect on Aß-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aß-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aß-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aß-induced impairments in hippocampal synaptic plasticity.

Fragile X mental retardation protein regulates heterosynaptic plasticity in the hippocampus.

Silencing of a single gene, FMR1, is linked to a highly prevalent form of mental retardation, characterized by social and cognitive impairments, known as fragile X syndrome (FXS). The FMR1 gene encodes fragile X mental retardation protein (FMRP), which negatively regulates translation. Knockout of Fmr1 in mice results in enhanced long-term depression (LTD) induced by metabotropic glutamate receptor (mGluR) activation. Despite the evidence implicating FMRP in LTD, the role of FMRP in long-term potentiation (LTP) is less clear. Synaptic strength can be augmented heterosynaptically through the generation and sequestration of plasticity-related proteins, in a cell-wide manner. If heterosynaptic plasticity is altered in Fmr1 knockout (KO) mice, this may explain the cognitive deficits associated with FXS. We induced homosynaptic plasticity using the ß-adrenergic receptor (ß-AR) agonist, isoproterenol (ISO), which facilitated heterosynaptic LTP that was enhanced in Fmr1 KO mice relative to wild-type (WT) controls. To determine if enhanced heterosynaptic LTP in Fmr1 KO mouse hippocampus requires protein synthesis, we applied a translation inhibitor, emetine (EME). EME blocked homo- and heterosynaptic LTP in both genotypes. We also probed the roles of mTOR and ERK in boosting heterosynaptic LTP in Fmr1 KO mice. Although heterosynaptic LTP was blocked in both WT and KOs by inhibitors of mTOR and ERK, homosynaptic LTP was still enhanced following mTOR inhibition in slices from Fmr1 KO mice. Because mTOR will normally stimulate translation initiation, our results suggest that ß-AR stimulation paired with derepression of translation results in enhanced heterosynaptic plasticity.