Fasudil treatment in adult reverses behavioural changes and brain ventricular enlargement in Oligophrenin-1 mouse model of intellectual disability.

Loss of function mutations in human Oligophrenin1 (OPHN1) gene are responsible for syndromic intellectual disability (ID) associated with cerebellar hypoplasia and cerebral ventricles enlargement. Functional studies in rodent models suggest that OPHN1 linked ID is a consequence of abnormal synaptic transmission and shares common pathophysiological mechanisms with other cognitive disorders. Variants of this gene have been also identified in autism spectrum disorder and schizophrenia. The advanced understanding of the mechanisms underlying OPHN1-related ID, allowed us to develop a therapeutic approach targeting the Ras homolog gene family, member A (RHOA) signalling pathway and repurpose Fasudil- a well-tolerated Rho Kinase (ROCK) and Protein Kinase A (PKA) inhibitor- as a treatment of ID. We have previously shown ex-vivo its beneficial effect on synaptic transmission and plasticity in a mouse model of the OPHN1 loss of function. Here, we report that chronic treatment in adult mouse with Fasudil, is able to counteract vertical and horizontal hyperactivities, restores recognition memory and limits the brain ventricular dilatation observed in Ophn1-/y However, deficits in working and spatial memories are partially or not rescued by the treatment. These results highlight the potential of Fasudil treatment in synaptopathies and also the need for multiple therapeutic approaches especially in adult where brain plasticity is reduced.

Coronin 1 regulates cognition and behavior through modulation of cAMP/protein kinase A signaling.

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic-AMP-protein kinase A-dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1-deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.

Altered microtubule dynamics in Mecp2-deficient astrocytes.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the gene MECP2 encoding the methyl-CpG binding protein 2. This genetic disease affects predominantly girls and is characterized by a period of normal development that lasts for 8-18 months, followed by neurologic regression affecting both motor and mental abilities. Previous studies performed on brains from RTT subjects and Mecp2-deficient mice showed striking changes in neuronal maturation and dendritic arborization. Recently, we showed that expression of stathmin-like 2 (STMN2) was significantly reduced in fibroblasts from RTT patients, and similar results were obtained in the cerebellum of Mecp2-deficient mice. Because assembly and dynamics of microtubules are known to be modulated by STMN2, we studied microtubule dynamics in brain cells from Mecp2-deficient mice. We observed that Mecp2 deficiency affects microtubule dynamics in astrocytes from Mecp2-deficient mice. Our data reinforce the fact that the loss of Mecp2 in astrocytes may influence the onset and progression of RTT. These results imply that Mecp2 has a stabilizing role in microtubule dynamics and that Mecp2 deficiency, which is associated with STMN2 down-regulation, could lead to impaired microtubule stability, hence explaining the dendritic abnormalities observed in RTT brains.

Chr21 protein-protein interactions: enrichment in proteins involved in intellectual disability, autism, and late-onset Alzheimer's disease.

Down syndrome (DS) is caused by human chromosome 21 (HSA21) trisomy. It is characterized by a poorly understood intellectual disability (ID). We studied two mouse models of DS, one with an extra copy of the <i>Dyrk1A</i> gene (189N3) and the other with an extra copy of the mouse Chr16 syntenic region (Dp(16)1Yey). RNA-seq analysis of the transcripts deregulated in the embryonic hippocampus revealed an enrichment in genes associated with chromatin for the 189N3 model, and synapses for the Dp(16)1Yey model. A large-scale yeast two-hybrid screen (82 different screens, including 72 HSA21 baits and 10 rebounds) of a human brain library containing at least 10⁷ independent fragments identified 1,949 novel protein-protein interactions. The direct interactors of HSA21 baits and rebounds were significantly enriched in ID-related genes (<i>P</i>-value &lt; 2.29 × 10^-8). Proximity ligation assays showed that some of the proteins encoded by HSA21 were located at the dendritic spine postsynaptic density, in a protein network at the dendritic spine postsynapse. We located HSA21 DYRK1A and DSCAM, mutations of which increase the risk of autism spectrum disorder (ASD) 20-fold, in this postsynaptic network. We found that an intracellular domain of DSCAM bound either DLGs, which are multimeric scaffolds comprising receptors, ion channels and associated signaling proteins, or DYRK1A. The DYRK1A-DSCAM interaction domain is conserved in <i>Drosophila</i> and humans. The postsynaptic network was found to be enriched in proteins associated with ARC-related synaptic plasticity, ASD, and late-onset Alzheimer's disease. These results highlight links between DS and brain diseases with a complex genetic basis.

Inhibition of RhoA pathway rescues the endocytosis defects in Oligophrenin1 mouse model of mental retardation.

The patho-physiological hypothesis of mental retardation caused by the deficiency of the RhoGAP Oligophrenin1 (OPHN1), relies on the well-known functions of Rho GTPases on neuronal morphology, i.e. dendritic spine structure. Here, we describe a new function of this Bin/Amphiphysin/Rvs domain containing protein in the control of clathrin-mediated endocytosis (CME). Through interactions with Src homology 3 domain containing proteins involved in CME, OPHN1 is concentrated to endocytic sites where it down-regulates the RhoA/ROCK signaling pathway and represses the inhibitory function of ROCK on endocytosis. Indeed disruption of Ophn1 in mice reduces the endocytosis of synaptic vesicles and the post-synaptic alpha-amino-3-hydroxy-5-methylisoazol-4-propionate (AMPA) receptor internalization, resulting in almost a complete loss of long-term depression in the hippocampus. Finally, pharmacological inhibition of this pathway by ROCK inhibitors fully rescued not only the CME deficit in OPHN1 null cells but also synaptic plasticity in the hippocampus from Ophn1 null model. Altogether, we uncovered a new patho-physiological mechanism for intellectual disabilities associated to mutations in RhoGTPases linked genes and also opened new directions for therapeutic approaches of congenital mental retardation.

IL1RAPL1 controls inhibitory networks during cerebellar development in mice.

Abnormalities in the formation and function of cerebellar circuitry potentially contribute to cognitive deficits in humans. In the adult, the activity of the sole output neurons of the cerebellar cortex - the Purkinje cells (PCs) - is shaped by the balance of activity between local excitatory and inhibitory circuits. However, how this balance is established during development remains poorly understood. Here, we investigate the role of interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), a protein linked to cognitive function which interacts with neuronal calcium sensor 1 (NCS-1) in the development of mouse cerebellum. Using Il1rapl1-deficient mice, we found that absence of IL1RAPL1 causes a transient disinhibition of deep cerebellar nuclei neurons between postnatal days 10 and 14 (P10/P14). Upstream, in the cerebellar cortex, we found developmental perturbations in the activity level of molecular layer interneurons (MLIs), resulting in the premature appearance of giant GABAA-mediated inhibitory post-synaptic currents capable of silencing PCs. Examination of feed-forward recruitment of MLIs by parallel fibres shows that during this P10/P14 time window, MLIs were more responsive to incoming excitatory drive. Thus, we conclude that IL1RAPL1 exerts a key function during cerebellar development in establishing local excitation/inhibition balance.

Loss of X-linked mental retardation gene oligophrenin1 in mice impairs spatial memory and leads to ventricular enlargement and dendritic spine immaturity.

Loss of oligophrenin1 (OPHN1) function in human causes X-linked mental retardation associated with cerebellar hypoplasia and, in some cases, with lateral ventricle enlargement. In vitro studies showed that ophn1 regulates dendritic spine through the control of Rho GTPases, but its in vivo function remains unknown. We generated a mouse model of ophn1 deficiency and showed that it mimics the ventricles enlargement without affecting the cerebellum morphoanatomy. The ophn1 knock-out mice exhibit behavioral defects in spatial memory together with impairment in social behavior, lateralization, and hyperactivity. Long-term potentiation and mGluR-dependent long-term depression are normal in the CA1 hippocampal area of ophn1 mutant, whereas paired-pulse facilitation is reduced. This altered short-term plasticity that reflects changes in the release of neurotransmitters from the presynaptic processes is associated with normal synaptic density together with a reduction in mature dendritic spines. In culture, inactivation of ophn1 function increases the density and proportion of immature spines. Using a conditional model of loss of ophn1 function, we confirmed this immaturity defect and showed that ophn1 is required at all the stages of the development. These studies show that, depending of the context, ophn1 controls the maturation of dendritic spines either by maintaining the density of mature spines or by limiting the extension of new filopodia. Altogether, these observations indicate that cognitive impairment related to OPHN1 loss of function is associated with both presynaptic and postsynaptic alterations.

Genetics and pathophysiology of mental retardation.

Mental retardation (MR) is defined as an overall intelligence quotient lower than 70, associated with functional deficit in adaptive behavior, such as daily-living skills, social skills and communication. Affecting 1-3% of the population and resulting from extraordinary heterogeneous environmental, chromosomal and monogenic causes, MR represents one of the most difficult challenges faced today by clinician and geneticists. Detailed analysis of the Online Mendelian Inheritance in Man database and literature searches revealed more than a thousand entries for MR, and more than 290 genes involved in clinical phenotypes or syndromes, metabolic or neurological disorders characterized by MR. We estimate that many more MR genes remain to be identified. The purpose of this review is to provide an overview on the remarkable progress achieved over the last decade in delineating genetic causes of MR, and to highlight the emerging biological and cellular processes and pathways underlying pathogeneses of human cognitive disorders.

Molecular cloning and expression pattern of the Fkbp25 gene during cerebral cortical neurogenesis.

Neocortical neurons are generated predominantly from the cells that proliferate in the ventricular zone of the telencephalon. In order to understand the nature of these expanding cortical neuronal progenitor cells, we selected by differential display some transcripts that were enriched in the telencephalon as compared to the more caudal regions (diencephalon/mesencephalon). This systematic screening revealed one of the differentially expressed transcripts, namely the Fkbp25 mRNA that encodes a member of the FK506 binding proteins (FKBPs). Northern blot analysis showed that the expression of the single 1.4kb Fkbp25 transcript reached a maximum level on embryonic day 11.5 at the start of cortical neurogenesis in the mouse and was followed by a weak basal expression in the adult brain. In the embryo, Fkbp25 gene was strongly expressed in the telencephalon ventricular zone but also in areas active in myogenesis (walls of the ventricle and the atrium) and chondrogenesis (the cartilage of the rib and the hindlimb). An increase in the transcript levels of the Fkbp25 gene was also observed during the two successive proliferation waves of the cerebellum development. Immunostaining on primary cultures of embryonic day 10.5 telencephalon stem cells showed that the Fkbp25 protein was present in the cytoplasm and nuclei of cells cultured for 6h but exclusively in the nuclei of the Tuj-1 immunoreactive neurons obtained after 3 days of culture (The sequence data reported here have been submitted to GenBank under accession no. AF135595.).

Early neuronal and glial determination from mouse E10.5 telencephalon embryonic stem cells: an in vitro study.

We report here a novel in vitro model for differentiating neuronal and glial cells from mouse embryonic day 10 telencephalon stem cells. At this developmental stage, the telencephalon consists of a single layer of neuroepithelial stem cells. We used various markers of proliferation and differentiation (Ki-67, nestin, BrdU, Tuj-1 and GFAP) to follow proliferative progenitors and to identify neuronal and glial derivatives. Neuronal derivatives were obtained from nestin+ progenitors. GFAP+ astrocytic derivatives were detected after only 72 h of culture. Both neuronal and glial derivatives were generated close to nestin-positive aggregates. In addition, we were able to manipulate neuronal determination of telencephalon stem cells by gene transient transfection as demonstrated by RP42 gene overexpression. These observations suggest that this in vitro model is of potential use for studying early steps in neuronal or glial determination from embryonic stem cells, an issue of key importance for adult brain cell therapy approaches.

Lack of the presynaptic RhoGAP protein oligophrenin1 leads to cognitive disabilities through dysregulation of the cAMP/PKA signalling pathway.

Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) lead to cognitive disabilities (CDs) in humans, yet the underlying mechanisms are not known. Here, we show that in mice constitutive lack of Ophn1 is associated with dysregulation of the cyclic adenosine monophosphate/phosphate kinase A (cAMP/PKA) signalling pathway in a brain-area-specific manner. Consistent with a key role of cAMP/PKA signalling in regulating presynaptic function and plasticity, we found that PKA-dependent presynaptic plasticity was completely abolished in affected brain regions, including hippocampus and amygdala. At the behavioural level, lack of OPHN1 resulted in hippocampus- and amygdala-related learning disabilities which could be fully rescued by the ROCK/PKA kinase inhibitor fasudil. Together, our data identify OPHN1 as a key regulator of presynaptic function and suggest that, in addition to reported postsynaptic deficits, loss of presynaptic plasticity contributes to the pathophysiology of CDs.

A circadian clock in hippocampus is regulated by interaction between oligophrenin-1 and Rev-erbα.

Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.

Synaptic maturation at cortical projections to the lateral amygdala in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a neuro-developmental disorder caused by loss of function of Mecp2--methyl-CpG-binding protein 2--an epigenetic factor controlling DNA transcription. In mice, removal of Mecp2 in the forebrain recapitulates most of behavioral deficits found in global Mecp2 deficient mice, including amygdala-related hyper-anxiety and lack of social interaction, pointing a role of Mecp2 in emotional learning. Yet very little is known about the establishment and maintenance of synaptic function in the adult amygdala and the role of Mecp2 in these processes. Here, we performed a longitudinal examination of synaptic properties at excitatory projections to principal cells of the lateral nucleus of the amygdala (LA) in Mecp2 mutant mice and their wild-type littermates. We first show that during animal life, Cortico-LA projections switch from a tonic to a phasic mode, whereas Thalamo-LA synapses are phasic at all ages. In parallel, we observed a specific elimination of Cortico-LA synapses and a decrease in their ability of generating presynaptic long term potentiation. In absence of Mecp2, both synaptic maturation and synaptic elimination were exaggerated albeit still specific to cortical projections. Surprisingly, associative LTP was unaffected at Mecp2 deficient synapses suggesting that synaptic maintenance rather than activity-dependent synaptic learning may be causal in RTT physiopathology. Finally, because the timing of synaptic evolution was preserved, we propose that some of the developmental effects of Mecp2 may be exerted within an endogenous program and restricted to synapses which maturate during animal life.

A postsynaptic signaling pathway that may account for the cognitive defect due to IL1RAPL1 mutation.

BACKGROUND: Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene mutations are associated with cognitive impairment ranging from nonsyndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of Toll/IL-1 receptors, whose expression in the brain is upregulated by neuronal activity. Currently, very little is known about the function of this protein. We previously showed that IL1RAPL1 interacts with the neuronal calcium sensor NCS-1 and that it regulates voltage-gated calcium channel activity in PC12 cells. RESULTS: Here we show that IL1RAPL1 is present in dendritic spine where it interacts with PSD-95, a major component of excitatory postsynaptic compartment. Using gain- and loss-of-function experiments in neurons, we demonstrated that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun terminal kinase (JNK) activity and PSD-95 phosphorylation. Mice carrying a null mutation of the mouse Il1rapl1 gene show a reduction of both dendritic spine density and excitatory synapses in the CA1 region of the hippocampus. These structural abnormalities are associated with specific deficits in hippocampal long-term synaptic plasticity. CONCLUSION: The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.

Role of mental retardation-associated dystrophin-gene product Dp71 in excitatory synapse organization, synaptic plasticity and behavioral functions.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. CONCLUSIONS/SIGNIFICANCE: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.