Defects in AMPAR trafficking and microglia activation underlie socio-cognitive deficits associated to decreased expression of phosphodiesterase 2 a.

Phosphodiesterase 2 A (PDE2A) is an enzyme involved in the homeostasis of cAMP and cGMP and is the most highly expressed PDE in human brain regions critical for socio-cognitive behavior. In cerebral cortex and hippocampus, PDE2A expression level is upregulated in Fmr1-KO mice, a model of the Fragile X Syndrome (FXS), the most common form of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Indeed, PDE2A translation is negatively modulated by FMRP, whose functional absence causes FXS. While the pharmacological inhibition of PDE2A has been associated to its pro-cognitive role in normal animals and in models of ID and ASD, homozygous PDE2A mutations have been identified in patients affected by ID, ASD and epilepsy. To clarify this apparent paradox about the role of PDE2A in brain development, we characterized here Pde2a+/- mice (homozygote animals being not viable) at the behavioral, cellular, molecular and electrophysiological levels. Pde2a+/- females display a milder form of the disorder with reduced cognitive performance in adulthood, conversely males show severe socio-cognitive deficits throughout their life. In males, these phenotypes are associated with microglia activation, elevated glutathione levels and increased externalization of Glutamate receptor (GluR1) in CA1, producing reduced mGluR-dependent Long-term Depression. Overall, our results reveal molecular targets of the PDE2A-dependent pathway underlying socio-cognitive performance. These results clarify the mechanism of action of pro-cognitive drugs based on PDE2A inactivation, which have been shown to be promising therapeutic approaches for Alzheimer's disease, schizophrenia, FXS as well as other forms of ASD.

Antilogic, a new supervised machine learning software for the automatic interpretation of antibiotic susceptibility testing in clinical microbiology: proof-of-concept on three frequently isolated bacterial species.

OBJECTIVE: Antibiotic susceptibility testing (AST) is necessary in order to adjust empirical antibiotic treatment, but the interpretation of results requires experience and knowledge. We have developed a machine learning software that is capable of reading AST images without any human intervention and that automatically interprets the AST, based on a database of antibiograms that have been clinically validated with European Committee on Antimicrobial Susceptibility Testing rules. METHODS: We built a database of antibiograms that were labelled by senior microbiologists for three species: Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. We then developed Antilogic, a Python software based on an original image segmentation module and supervised learning models that we trained against the database. Finally, we blind tested Antilogic against a validation set of 5100 photos of antibiograms. RESULTS: We trained Antilogic against a database of 18072 pictures of antibiograms. Overall agreement against the validation set reached 97% (16 855/17 281) regarding phenotypes. The severity rate of errors was also evaluated: 1.66% (287/17 281) were major errors and 0.80% (136/17 281) were very major errors. After implementation of uncertainty quantifications, the rate of errors decreased to 0.80% (114/13 451) and 0.42% (51/13 451) for major and very major errors respectively. DISCUSSION: Antilogic is the first machine learning software that has been developed for AST interpretation. It is based on a novel approach that differs from the typical diameter measurement and expert system approach. Antilogic is a proof of concept that artificial intelligence can contribute to faster and easier diagnostic methods in the field of clinical microbiology.

Agonist-induced functional analysis and cell sorting associated with single-cell transcriptomics characterizes cell subtypes in normal and pathological brain.

To gain better insight into the dynamic interaction between cells and their environment, we developed the agonist-induced functional analysis and cell sorting (aiFACS) technique, which allows the simultaneous recording and sorting of cells in real-time according to their immediate and individual response to a stimulus. By modulating the aiFACS selection parameters, testing different developmental times, using various stimuli, and multiplying the analysis of readouts, it is possible to analyze cell populations of any normal or pathological tissue. The association of aiFACS with single-cell transcriptomics allows the construction of functional tissue cartography based on specific pharmacological responses of cells. As a proof of concept, we used aiFACS on the dissociated mouse brain, a highly heterogeneous tissue, enriching it in interneurons by stimulation with KCl or with AMPA, an agonist of the glutamate receptors, followed by sorting based on calcium levels. After AMPA stimulus, single-cell transcriptomics of these aiFACS-selected interneurons resulted in a nine-cluster classification. Furthermore, we used aiFACS on interneurons derived from the brain of the Fmr1-KO mouse, a rodent model of fragile X syndrome. We showed that these interneurons manifest a generalized defective response to AMPA compared with wild-type cells, affecting all the analyzed cell clusters at one specific postnatal developmental time.

Reduction of Fmr1 mRNA Levels Rescues Pathological Features in Cortical Neurons in a Model of FXTAS.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55-200 CGG repeat expansion in the 5' UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches.

Involvement of Phosphodiesterase 2A Activity in the Pathophysiology of Fragile X Syndrome.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in translational regulation of mRNAs that play key roles in synaptic morphology and plasticity. The functional absence of FMRP causes the fragile X syndrome (FXS), the most common form of inherited intellectual disability and the most common monogenic cause of autism. No effective treatment is available for FXS. We recently identified the Phosphodiesterase 2A (Pde2a) mRNA as a prominent target of FMRP. PDE2A enzymatic activity is increased in the brain of Fmr1-KO mice, a recognized model of FXS, leading to decreased levels of cAMP and cGMP. Here, we pharmacologically inhibited PDE2A in Fmr1-KO mice and observed a rescue both of the maturity of dendritic spines and of the exaggerated hippocampal mGluR-dependent long-term depression. Remarkably, PDE2A blockade rescued the social and communicative deficits of both mouse and rat Fmr1-KO animals. Importantly, chronic inhibition of PDE2A in newborn Fmr1-KO mice followed by a washout interval, resulted in the rescue of the altered social behavior observed in adolescent mice. Altogether, these results reveal the key role of PDE2A in the physiopathology of FXS and suggest that its pharmacological inhibition represents a novel therapeutic approach for FXS.

New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and a leading cause of autism, results from the loss of expression of the Fmr1 gene which encodes the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Among the thousands mRNA targets of FMRP, numerous encode regulators of ion homeostasis. It has also been described that FMRP directly interacts with Ca2+ channels modulating their activity. Collectively these findings suggest that FMRP plays critical roles in Ca2+ homeostasis during nervous system development. We carried out a functional analysis of Ca2+ regulation using a calcium imaging approach in Fmr1-KO cultured neurons and we show that these cells display impaired steady state Ca2+ concentration and an altered entry of Ca2+ after KCl-triggered depolarization. Consistent with these data, we show that the protein product of the Cacna1a gene, the pore-forming subunit of the Cav2.1 channel, is less expressed at the plasma membrane of Fmr1-KO neurons compared to wild-type (WT). Thus, our findings point out the critical role that Cav2.1 plays in the altered Ca2+ flux in Fmr1-KO neurons, impacting Ca2+ homeostasis of these cells. Remarkably, we highlight a new phenotype of cultured Fmr1-KO neurons that can be considered a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

Exploration and characterisation of the phenotypic and genetic profiles of patients with early onset schizophrenia associated with autism spectrum disorder and their first-degree relatives: a French multicentre case series study protocol (GenAuDiss).

INTRODUCTION: Early-onset schizophrenia (EOS) is a rare and severe condition. A higher rate of neurodevelopmental abnormalities, such as intellectual or communication impairments as well as attention deficit hyperactivity disorder, is observed in EOS compared with adult-onset schizophrenia. Early signs of autism spectrum disorders (ASD) are present in about 30% of patients. Genetic abnormalities, including copy number variations, are frequent in neurodevelopmental disorders and have been associated to ASD physiopathology. Implicated genes encode proteins involved in brain development, synapses morphology and plasticity and neurogenesis. In addition, an increasing number of genetic abnormalities are shared by EOS and ASD, underlying the neurodevelopmental hypothesis of EOS.The main objective of our study is to identify disease-causing genetic mutations in a cohort of patients affected by both EOS and ASD. Special attention will be paid to genes involved in neurodevelopmental pathways. METHODS AND ANALYSIS: We describe a multicentric study in a paediatric population. The study started in April 2014. Inclusion criteria are: age 7-22 years, diagnosis of EOS with comorbid ASD and IQ >50; Parents and siblings are also enrolled. We perform psychiatric assessments (Mini International Neuropsychiatric Interview, Kiddie Schedule for Affective Disorders and Schizophrenia -Present and Lifetime Version, Positive and Negative Syndrome Scale and Scale for the Assessment of Negative Symptoms) together with neurocognitive evaluations (IQ, Trail Making Test A/B and verbal fluency). Then, we study variants of the coding part of DNA (exome), using next-generation sequencing process on trio (mother, father and child). Bioinformatics tools (RVIS and PolyPhen-2) are used to prioritise disease-causing mutations in candidate genes. The inclusion period will end in November 2019. ETHICS AND DISSEMINATION: The study protocol was approved by the Local Ethic Committee and by the French National Agency for Medicines and Health Products Safety. All patients signed informed consent on enrolment in the study. Results of the present study should help to unravel the molecular pathology of EOS, paving the way for an early therapeutic intervention. TRIAL REGISTRATION NUMBER: NCT0256552; Pre-results.

HITS-CLIP in various brain areas reveals new targets and new modalities of RNA binding by fragile X mental retardation protein.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson-Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.

Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

The Search for an Effective Therapy to Treat Fragile X Syndrome: Dream or Reality?

Fragile X Syndrome (FXS) is the most common form of intellectual disability and a primary cause of autism. It originates from the lack of the Fragile X Mental Retardation Protein (FMRP), which is an RNA-binding protein encoded by the Fragile X Mental Retardation Gene 1 (FMR1) gene. Multiple roles have been attributed to this protein, ranging from RNA transport (from the nucleus to the cytoplasm, but also along neurites) to translational control of mRNAs. Over the last 20 years many studies have found a large number of FMRP mRNA targets, but it is still not clear which are those playing a critical role in the etiology of FXS. So far, no therapy for FXS has been found, making the quest for novel targets of considerable importance. Several pharmacological approaches have been attempted, but, despite some promising preclinical results, no strategy gave successful outcomes, due either to the induction of major side effects or to the lack of improvement of the phenotypes. However, these studies suggested that, in order to measure the effectiveness of a specific treatment, trials should be redesigned and new endpoints defined in FXS patients. Nevertheless, the search for new therapeutic targets for FXS is very active. In this context, the advances in animal modeling, coupled with better understanding of neurobiology and physiopathology of FXS, are of crucial importance in developing new selected treatments. Here, we discuss the pathways that were recently linked to the physiopathology of FXS (mGluR, GABAR, insulin, Insulin-like Growth Factor 1 (IGF-1), MPP-9, serotonin, oxytocin and endocannabinoid signaling) and that suggest new approaches to find an effective therapy for this disorder. Our goal with this review article is to summarize some recent relevant findings on FXS treatment strategies in order to have a clearer view of the different pathways analyzed to date emphasizing those shared with other synaptic disorders.

A new cis-acting motif is required for the axonal SMN-dependent Anxa2 mRNA localization.

Spinal muscular atrophy (SMA) is caused by mutations and/or deletions of the survival motor neuron gene (SMN1). Besides its function in the biogenesis of spliceosomal snRNPs, SMN might possess a motor neuron specific role and could function in the transport of axonal mRNAs and in the modulation of local protein translation. Accordingly, SMN colocalizes with axonal mRNAs of differentiated NSC-34 motor neuron-like cells. We recently showed that SMN depletion gives rise to a decrease in the axonal transport of the mRNAs encoding Annexin A2 (Anxa2). In this work, we have characterized the structural features of the Anxa2 mRNA required for its axonal targeting by SMN. We found that a G-rich motif located near the 3'UTR is essential for axonal localization of the Anxa2 transcript. We also show that mutations in the motif sequence abolish targeting of Anxa2 reporter mRNAs in axon-like structures of differentiated NSC-34 cells. Finally, localization of both wild-type and mutated Anxa2 reporters is restricted to the cell body in SMN-depleted cells. Altogether, our studies show that this G-motif represents a novel and essential determinant for axonal localization of the Anxa2 mRNA mediated by the SMN complex.

New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes.

Cytoplasmic FMRP interacting protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene), whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC), thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models - fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction.

The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.

Fragile X Syndrome: from molecular pathology to therapy.

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability due to the silencing of the FMR1 gene encoding FMRP (Fragile X Mental Retardation Protein), an RNA-binding protein involved in different steps of RNA metabolism. Of particular interest is the key role of FMRP in translational regulation. Since the first functional characterizations of FMRP, its role has been underlined by its association with actively translating polyribosomes. Furthermore, a plethora of mRNA targets of FMRP have been identified. In the absence of FMRP the deregulation of translation/transport/stability of these mRNAs has a cascade effect on many pathways, resulting into the final phenotype. We review here a set of targets of FMRP (mRNAs and proteins) that may have an impact on the FXS phenotype by deregulating some key cellular processes, such as translation, cytoskeleton remodeling and oxidative stress. The manipulation of these abnormal pathways by specific drugs may represent new therapeutic opportunities for FXS patients.

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila.

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼ 23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.

Anesthesia-induced hypothermia mediates decreased ARC gene and protein expression through ERK/MAPK inactivation.

Several anesthetics have been reported to suppress the transcription of a number of genes, including Arc, also known as Arg3.1, an immediate early gene that plays a significant role in memory consolidation. The purpose of this study was to explore the mechanism of anesthesia-mediated depression in Arc gene and protein expression. Here, we demonstrate that isoflurane or propofol anesthesia decreases hippocampal Arc protein expression in rats and mice. Surprisingly, this change was secondary to anesthesia-induced hypothermia. Furthermore, we confirm in vivo and in vitro that hypothermia per se is directly responsible for decreased Arc protein levels. This effect was the result of the decline of Arc mRNA basal levels following inhibition of ERK/MAPK by hypothermia. Overall, our results suggest that anesthesia-induced hypothermia leads to ERK inhibition, which in turns decreases Arc levels. These data give new mechanistic insights on the regulation of immediate early genes by anesthesia and hypothermia.

RNase III-independent microRNA biogenesis in mammalian cells.

RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, we describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. We provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes.

Functional parameters of Dicer-independent microRNA biogenesis.

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.