Dissecting the contribution of host genetics and the microbiome in complex behaviors.

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.

Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring.

Maternal obesity during pregnancy has been associated with increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD), in offspring. Here, we report that maternal high-fat diet (MHFD) induces a shift in microbial ecology that negatively impacts offspring social behavior. Social deficits and gut microbiota dysbiosis in MHFD offspring are prevented by co-housing with offspring of mothers on a regular diet (MRD) and transferable to germ-free mice. In addition, social interaction induces synaptic potentiation (LTP) in the ventral tegmental area (VTA) of MRD, but not MHFD offspring. Moreover, MHFD offspring had fewer oxytocin immunoreactive neurons in the hypothalamus. Using metagenomics and precision microbiota reconstitution, we identified a single commensal strain that corrects oxytocin levels, LTP, and social deficits in MHFD offspring. Our findings causally link maternal diet, gut microbial imbalance, VTA plasticity, and behavior and suggest that probiotic treatment may relieve specific behavioral abnormalities associated with neurodevelopmental disorders. VIDEO ABSTRACT.

Suppression of PKR promotes network excitability and enhanced cognition by interferon-γ-mediated disinhibition.

The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr(-/-) phenotype in WT mice, enhancing long-term memory storage and L-LTP.

eIF2α controls memory consolidation via excitatory and somatostatin neurons.

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP.

Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.

A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping.

BACKGROUND: The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. RESULTS: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. CONCLUSIONS: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Translational control in synaptic plasticity and cognitive dysfunction.

Activity-dependent changes in the strength of synaptic connections are fundamental to the formation and maintenance of memory. The mechanisms underlying persistent changes in synaptic strength in the hippocampus, specifically long-term potentiation and depression, depend on new protein synthesis. Such changes are thought to be orchestrated by engaging the signaling pathways that regulate mRNA translation in neurons. In this review, we discuss the key regulatory pathways that govern translational control in response to synaptic activity and the mRNA populations that are specifically targeted by these pathways. The critical contribution of regulatory control over new protein synthesis to proper cognitive function is underscored by human disorders associated with either silencing or mutation of genes encoding proteins that directly regulate translation. In light of these clinical implications, we also consider the therapeutic potential of targeting dysregulated translational control to treat cognitive disorders of synaptic dysfunction.

RAPping production of type I interferon in pDCs through mTOR.

The production of type I interferon -- the first line of defense against virus infection and critical for innate immunity -- in plasmacytoid dendritic cells relies on the mammalian target of rapamycin.

Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal-dependent cognitive deficits induced by TBI in two different injury mouse models-focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma-induced chronic cognitive deficits.

Postnatal deamidation of 4E-BP2 in brain enhances its association with raptor and alters kinetics of excitatory synaptic transmission.

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Repeated mild traumatic brain injury produces neuroinflammation, anxiety-like behaviour and impaired spatial memory in mice.

PRIMARY OBJECTIVE: Repeated traumatic brain injuries (rmTBI) are frequently associated with debilitating neuropsychiatric conditions such as cognitive impairment, mood disorders, and post-traumatic stress disorder. We tested the hypothesis that repeated mild traumatic brain injury impairs spatial memory and enhances anxiety-like behaviour. RESEARCH DESIGN: We used a between groups design using single (smTBI) or repeated (rmTBI) controlled cranial closed skull impacts to mice, compared to a control group. METHODS AND PROCEDURES: We assessed the effects of smTBI and rmTBI using measures of motor performance (Rotarod Test [RT]), anxiety-like behaviour (Elevated Plus Maze [EPM] and Open Field [OF] tests), and spatial memory (Morris Water Maze [MWM]) within 12 days of the final injury. In separate groups of mice, astrocytosis and microglial activation were assessed 24 hours after the final injury using GFAP and IBA-1 immunohistochemistry. MAIN OUTCOMES AND RESULTS: RmTBI impaired spatial memory in the MWM and increased anxiety-like behaviour in the EPM and OFT. In addition, rmTBI elevated GFAP and IBA-1 immunohistochemistry throughout the mouse brain. RmTBI produced astrocytosis and microglial activation, and elicited impaired spatial memory and anxiety-like behaviour. CONCLUSIONS: rmTBI produces acute cognitive and anxiety-like disturbances associated with inflammatory changes in brain regions involved in spatial memory and anxiety.

Rett syndrome like phenotypes in the R255X Mecp2 mutant mouse are rescued by MECP2 transgene.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that is usually caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). Four of the eight common disease causing mutations in MECP2 are nonsense mutations and are responsible for over 35% of all cases of RTT. A strategy to overcome disease-causing nonsense mutations is treatment with nonsense mutation suppressing drugs that allow expression of full-length proteins from mutated genes with premature in-frame stop codons. To determine if this strategy is useful in RTT, we characterized a new mouse model containing a knock-in nonsense mutation (p.R255X) in the Mecp2 locus (Mecp2(R255X)). To determine whether the truncated gene product acts as a dominant negative allele and if RTT-like phenotypes could be rescued by expression of wild-type protein, we genetically introduced an extra copy of MECP2 via an MECP2 transgene. The addition of MECP2 transgene to Mecp2(R255X) mice abolished the phenotypic abnormalities and resulted in near complete rescue. Expression of MECP2 transgene Mecp2(R255X) allele also rescued mTORC1 signaling abnormalities discovered in mice with loss of function and overexpression of Mecp2. Finally, we treated Mecp2(R255X) embryonic fibroblasts with the nonsense mutation suppressing drug gentamicin and we were able to induce expression of full-length MeCP2 from the mutant p.R255X allele. These data provide proof of concept that the p.R255X mutation of MECP2 is amenable to the nonsense suppression therapeutic strategy and provide guidelines for the extent of rescue that can be expected by re-expressing MeCP2 protein.

Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism.

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.

Truncation of Ube3a-ATS unsilences paternal Ube3a and ameliorates behavioral defects in the Angelman syndrome mouse model.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.

Translational control of the innate immune response through IRF-7.

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-alpha and IFN-beta), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Selective pharmacogenetic inhibition of mammalian target of Rapamycin complex I (mTORC1) blocks long-term synaptic plasticity and memory storage.

Both the formation of long-term memory (LTM) and late-long-term potentiation (L-LTP), which is thought to represent the cellular model of learning and memory, require de novo protein synthesis. The mammalian target of Rapamycin (mTOR) complex I (mTORC1) integrates information from various synaptic inputs and its best characterized function is the regulation of translation. Although initial studies have shown that rapamycin reduces L-LTP and partially blocks LTM, recent genetic and pharmacological evidence indicating that mTORC1 promotes L-LTP and LTM is controversial. Thus, the role of mTORC1 in L-LTP and LTM is unclear. To selectively inhibit mTORC1 activity in the adult brain, we used a "pharmacogenetic" approach that relies on the synergistic action of a drug (rapamycin) and a genetic manipulation (mTOR heterozygotes, mTOR(+/-) mice) on the same target (mTORC1). Although L-LTP and LTM are normal in mTOR(+/-) mice, application of a low concentration of rapamycin-one that is subthreshold for WT mice-prevented L-LTP and LTM only in mTOR(+/-) mice. Furthermore, we found that mTORC1-mediated translational control is required for memory reconsolidation. We provide here direct genetic evidence supporting the role of mTORC1 in L-LTP and behavioral memory.

Persistent ∆FosB expression limits recurrent seizure activity and provides neuroprotection in the dentate gyrus of APP mice.

Recurrent seizures lead to accumulation of the activity-dependent transcription factor ∆FosB in hippocampal dentate granule cells in both mouse models of epilepsy and mouse models of Alzheimer's disease (AD), which is also associated with increased incidence of seizures. In patients with AD and related mouse models, the degree of ∆FosB accumulation corresponds with increasing severity of cognitive deficits. We previously found that ∆FosB impairs spatial memory in mice by epigenetically regulating expression of target genes such as calbindin that are involved in synaptic plasticity. However, the suppression of calbindin in conditions of neuronal hyperexcitability has been demonstrated to provide neuroprotection to dentate granule cells, indicating that ∆FosB may act over long timescales to coordinate neuroprotective pathways. To test this hypothesis, we used viral-mediated expression of ∆JunD to interfere with ∆FosB signaling over the course of several months in transgenic mice expressing mutant human amyloid precursor protein (APP), which exhibit spontaneous seizures and develop AD-related neuropathology and cognitive deficits. Our results demonstrate that persistent ∆FosB activity acts through discrete modes of hippocampal target gene regulation to modulate neuronal excitability, limit recurrent seizure activity, and provide neuroprotection to hippocampal dentate granule cells in APP mice.

Precision microbial intervention improves social behavior but not autism severity: A pilot double-blind randomized placebo-controlled trial.

Autism spectrum disorder (ASD) is characterized by the presence of restricted/repetitive behaviors and social communication deficits. Because effective treatments for ASD remain elusive, novel therapeutic strategies are necessary. Preclinical studies show that L. reuteri selectively reversed social deficits in several models for ASD. Here, in a double-blind, randomized, placebo-controlled trial, we tested the effect of L. reuteri (a product containing a combination of strains ATCC-PTA-6475 and DSM-17938) in children with ASD. The treatment does not alter overall autism severity, restricted/repetitive behaviors, the microbiome composition, or the immune profile. However, L. reuteri combination yields significant improvements in social functioning that generalized across different measures. Interestingly, ATCC-PTA-6475, but not the parental strain of DSM-17938, reverses the social deficits in a preclinical mouse model for ASD. Collectively, our findings show that L. reuteri enhances social behavior in children with ASD, thereby warranting larger trials in which strain-specific effects should also be investigated.

Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production.

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSV(DeltaM51)), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV(DeltaM51) selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSV(DeltaM51).