Dendritic spine instability and insensitivity to modulation by sensory experience in a mouse model of fragile X syndrome.
ABSTRACT: Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by transcriptional inactivation of the X-linked fragile X mental retardation 1 (FMR1) gene. FXS is associated with increased density and abnormal morphology of dendritic spines, the postsynaptic sites of the majority of excitatory synapses. To better understand how lack of the FMR1 gene function affects spine development and plasticity, we examined spine formation and elimination of layer 5 pyramidal neurons in the whisker barrel cortex of Fmr1 KO mice with a transcranial two-photon imaging technique. We found that the rates of spine formation and elimination over days to weeks were significantly higher in both young and adult KO mice compared with littermate controls. The heightened spine turnover in KO mice was due to the existence of a larger pool of "short-lived" new spines in KO mice than in controls. Furthermore, we found that the formation of new spines and the elimination of existing ones were less sensitive to modulation by sensory experience in KO mice. These results indicate that the loss of Fmr1 gene function leads to ongoing overproduction of transient spines in the primary somatosensory cortex. The insensitivity of spine formation and elimination to sensory alterations in Fmr1 KO mice suggest that the developing synaptic circuits may not be properly tuned by sensory stimuli in FXS.
Project description:Fragile X Syndrome (FXS) is the major cause of inherited mental retardation and the leading genetic cause of Autism spectrum disorders. FXS is caused by mutations in the Fragile X Mental Retardation 1 (Fmr1) gene, which results in transcriptional silencing of Fragile X Mental Retardation Protein (FMRP). To elucidate cellular mechanisms involved in the pathogenesis of FXS, we compared dendritic spines in the hippocampal CA1 region of adult wild-type (WT) and Fmr1 knockout (Fmr1-KO) mice. Using diolistic labeling, confocal microscopy, and three-dimensional electron microscopy, we show a significant increase in the diameter of secondary dendrites, an increase in dendritic spine density, and a decrease in mature dendritic spines in adult Fmr1-KO mice. While WT and Fmr1-KO mice had the same mean density of spines, the variance in spine density was three times greater in Fmr1-KO mice. Reduced astrocyte participation in the tripartite synapse and less mature post-synaptic densities were also found in Fmr1-KO mice. We investigated whether the increase in synaptic spine density was associated with altered synaptic pruning during development. Our data are consistent with reduced microglia-mediated synaptic pruning in the CA1 region of Fmr1-KO hippocampi when compared with WT littermates at postnatal day 21, which is the peak period of synaptic pruning in the mouse hippocampus. Collectively, these results support abnormal synaptogenesis and synaptic remodeling in mice deficient in FMRP. Deficits in the maturation and distribution of synaptic spines on dendrites of CA1 hippocampal neurons may play a role in the intellectual disabilities associated with FXS.
Project description:Fragile X syndrome (FXS) is the most common type of mental retardation attributable to a single-gene mutation. It is caused by FMR1 gene silencing and the consequent loss of its protein product, fragile X mental retardation protein. Fmr1 global knockout (KO) mice recapitulate many behavioral and synaptic phenotypes associated with FXS. Abundant evidence suggests that astrocytes are important contributors to neurological diseases. This study investigates astrocytic contributions to the progression of synaptic abnormalities and learning impairments associated with FXS.Taking advantage of the Cre-lox system, we generated and characterized mice in which fragile X mental retardation protein is selectively deleted or exclusively expressed in astrocytes. We performed in vivo two-photon imaging to track spine dynamics/morphology along dendrites of neurons in the motor cortex and examined associated behavioral defects.We found that adult astrocyte-specific Fmr1 KO mice displayed increased spine density in the motor cortex and impaired motor-skill learning. The learning defect coincided with a lack of enhanced spine dynamics in the motor cortex that normally occurs in response to motor skill acquisition. Although spine density was normal at 1 month of age in astrocyte-specific Fmr1 KO mice, new spines formed at an elevated rate. Furthermore, fragile X mental retardation protein expression in only astrocytes was insufficient to rescue most spine or behavioral defects.Our work suggests a joint astrocytic-neuronal contribution to FXS pathogenesis and reveals that heightened spine formation during adolescence precedes the overabundance of spines and behavioral defects found in adult Fmr1 KO mice.
Project description:Structural dynamics of dendritic spines are important for memory and learning and are impaired in neurodevelopmental disorders such as fragile X syndrome. Spine dynamics are regulated by activity-dependent mechanisms that involve modulation of AMPA receptors (AMPAR); however, the relationship between AMPAR and spine dynamics in vivo and how these are altered in FXS mouse model is not known. Here, we tracked AMPAR and spines over multiple days in vivo in the cortex and found that dendritic spines in the fmr1 KO mouse were denser, smaller, had higher turnover rates and contained less sGluA2 compared to littermate controls. Although, KO spines maintained the relationship between AMPAR and spine stability, AMPAR levels in the KO were more dynamic with larger proportion of spines showing multiple dynamic events of AMPAR. Directional changes in sGluA2 were also observed in newly formed and eliminated spines, with KO spines displaying greater loss of AMPAR before elimination. Thus, we demonstrate that AMPAR levels within spines not are only continuously dynamic, but are also predictive of spine behavior, with impairments observed in the fmr1 KO mice.
Project description:Fragile X syndrome (FXS) is the most common inherited form of autism and intellectual disability and is caused by the silencing of a single gene, fragile X mental retardation 1 (Fmr1). The Fmr1 KO mouse displays phenotypes similar to symptoms in the human condition--including hyperactivity, repetitive behaviors, and seizures--as well as analogous abnormalities in the density of dendritic spines. Here we take a hypothesis-driven, mechanism-based approach to the search for an effective therapy for FXS. We hypothesize that a treatment that rescues the dendritic spine defect in Fmr1 KO mice may also ameliorate autism-like behavioral symptoms. Thus, we targeted a protein that regulates spines through modulation of actin cytoskeleton dynamics: p21-activated kinase (PAK). Our results demonstrate that a potent small molecule inhibitor of group I PAKs reverses dendritic spine phenotypes in Fmr1 KO mice. Moreover, this PAK inhibitor--which we call FRAX486--also rescues seizures and behavioral abnormalities such as hyperactivity and repetitive movements, thereby supporting the hypothesis that a drug treatment that reverses the spine abnormalities can also treat neurological and behavioral symptoms. Finally, a single administration of FRAX486 is sufficient to rescue all of these phenotypes in adult Fmr1 KO mice, demonstrating the potential for rapid, postdiagnostic therapy in adults with FXS.
Project description:Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by silencing of the FMR1 gene and subsequent loss of its protein product, fragile X retardation protein (FMRP). One of the most robust neuropathological findings in post-mortem human FXS and Fmr1 KO mice is the abnormal increase in dendritic spine densities, with the majority of spines showing an elongated immature morphology. However, the exact mechanisms of how FMRP can regulate dendritic spine development are still unclear. Abnormal dendritic spines can result from disturbances of multiple factors during neurodevelopment, such as alterations in neuron numbers, position and glial cells. In this study, we undertook a comprehensive histological analysis of the cerebral cortex in Fmr1 KO mice. They displayed significantly fewer neuron and PV-interneuron numbers, along with altered cortical lamination patterns. In terms of glial cells, Fmr1 KO mice exhibited an increase in Olig2-oligodendrocytes, which corresponded to the abnormally higher myelin expression in the corpus callosum. Iba1-microglia were significantly reduced but GFAP-astrocyte numbers and intensity were elevated. Using primary astrocytes derived from KO mice, we further demonstrated the presence of astrogliosis characterized by an increase in GFAP expression and astrocyte hypertrophy. Our findings provide important information on the cortical architecture of Fmr1 KO mice, and insights towards possible mechanisms associated with FXS.
Project description:Fragile X syndrome (FXS) is a neurodevelopmental disorder that causes intellectual disability, as well as the leading monogenic cause of autism spectrum disorders (ASD), in which neurons show aberrant dendritic spine structure. The reduction/absence of the functional FMRP protein, coded by the X-linked Fmr1 gene in humans, is responsible for the syndrome. Targets of FMRP, CLSTN1, and ICAM5, play critical roles in the maturation of dendritic spines, synapse formation and synaptic plasticity. However, the implication of CLSTN1 and ICAM5 in dendritic spine abnormalities and the underlying neuropathologic processes in FXS remain uninvestigated. In this study, we demonstrated that CLSTN1 co-localizes and co-transports with ICAM5 in cultured cortical neurons. Also we showed that shRNA-mediated downregulation of CLSTN1 in cultured WT neurons increases ICAM5 on the surface of synaptic membrane, subsequently affecting the maturation of dendritic spines. Whereas, normalization of CLSTN1 level in Fmr1 KO neurons reduces ICAM5 abundance and rescues impaired dendritic spine phenotypes. Most importantly, CLSTN1 protein is reduced in the postnatal medial prefrontal cortex of Fmr1 KO mice, which is correlated with increased ICAM5 levels on the surface of synapses and excessive filopodia-like spines. In conclusion, this study demonstrates that CLSTN1 plays a critical role in dendritic spine formation and maturation in FXS by regulating ICAM5 redistribution.
Project description:Fragile X syndrome (FXS) causes mental impairment and autism through transcriptional silencing of the Fmr1 gene, resulting in the loss of the RNA-binding protein fragile X mental retardation protein (FMRP). Cortical pyramidal neurons in affected individuals and Fmr1 knock-out (KO) mice have an increased density of dendritic spines. The mutant mice also show defects in synaptic and experience-dependent circuit plasticity, which are known to be mediated in part by dendritic spine dynamics. We used in vivo time-lapse imaging with two-photon microscopy through cranial windows in male and female neonatal mice to test the hypothesis that dynamics of dendritic protrusions are altered in KO mice during early postnatal development. We find that layer 2/3 neurons from wild-type mice exhibit a rapid decrease in dendritic spine dynamics during the first 2 postnatal weeks, as immature filopodia are replaced by mushroom spines. In contrast, KO mice show a developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes. Blockade of metabotropic glutamate receptor (mGluR) signaling, which reverses some adult phenotypes of KO mice, accentuated this immature protrusion phenotype in KO mice. Thus, absence of FMRP delays spine stabilization and dysregulated mGluR signaling in FXS may partially normalize this early synaptic defect.
Project description:Fragile X syndrome (FXS), the most commonly inherited form of mental retardation and autism, is caused by transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene and consequent loss of the fragile X mental retardation protein. Despite growing evidence suggesting a role of specific receptors and biochemical pathways in FXS pathogenesis, an effective therapeutic method has not been developed. Here, we report that abnormalities in FMR1 knockout (KO) mice, an animal model of FXS, are ameliorated, at least partially, at both cellular and behavioral levels, by an inhibition of the catalytic activity of p21-activated kinase (PAK), a kinase known to play a critical role in actin polymerization and dendritic spine morphogenesis. Greater spine density and elongated spines in the cortex, morphological synaptic abnormalities commonly observed in FXS, are at least partially restored by postnatal expression of a dominant negative (dn) PAK transgene in the forebrain. Likewise, the deficit in cortical long-term potentiation observed in FMR1 KO mice is fully restored by the dnPAK transgene. Several behavioral abnormalities associated with FMR1 KO mice, including those in locomotor activity, stereotypy, anxiety, and trace fear conditioning are also ameliorated, partially or fully, by the dnPAK transgene. Finally, we demonstrate a direct interaction between PAK and fragile X mental retardation protein in vitro. Overall, our results demonstrate the genetic rescue of phenotypes in a FXS mouse model and suggest that the PAK signaling pathway, including the catalytic activity of PAK, is a novel intervention site for development of an FXS and autism therapy.
Project description:Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The neuroanatomical phenotype of adult FXS patients, as well as adult Fmr1 knockout (KO) mice, includes elevated dendritic spine density and a spine morphology profile in neocortex that resembles younger individuals. Developmental studies in mouse neocortex have revealed a dynamic phenotype that varies with age, especially during the period of synaptic pruning. Here we investigated the hippocampal dentate gyrus to determine if the FXS spine phenotype is similarly tied to periods of maturation and pruning in this brain region. We used high-voltage electron microscopy to characterize Golgi-stained spines along granule cell dendrites in Fmr1 KO and wildtype (WT) mouse dentate gyrus at postnatal days 15, 21, 30, and 60. In contrast to neocortex, dendritic spine density was higher in Fmr1 KO mice across development. Interestingly, neither genotype showed specific phases of synaptogenesis or pruning, potentially explaining the phenotypic differences from neocortex. Similarly, although the KO mice showed a more immature morphological phenotype overall than WT (higher proportion of thin headed spines, lower proportion of mushroom and stubby spines), both genotypes showed gradual development, rather than impairments during specific phases of maturation. Finally, spine length showed a complex developmental pattern that differs from other brain regions examined, suggesting dynamic regulation by FMRP and other brain region-specific proteins. These findings shed new light on FMRP's role in development and highlight the need for new techniques to further understand the mechanisms by which FMRP affects synaptic maturation.
Project description:Fragile X syndrome (FXS) is the most common inherited cause of intellectual disabilities and a leading cause of autism. FXS is caused by a trinucleotide expansion in the gene FMR1 on the X chromosome. The neuroanatomical hallmark of FXS is an overabundance of immature dendritic spines, a factor thought to underlie synaptic dysfunction and impaired cognition. We showed that aberrantly increased activity of the Rho GTPase Rac1 inhibited the actin-depolymerizing factor cofilin, a major determinant of dendritic spine structure, and caused disease-associated spine abnormalities in the somatosensory cortex of FXS model mice. Increased cofilin phosphorylation and actin polymerization coincided with abnormal dendritic spines and impaired synaptic maturation. Viral delivery of a constitutively active cofilin mutant (cofilinS3A) into the somatosensory cortex of Fmr1-deficient mice rescued the immature dendritic spine phenotype and increased spine density. Inhibition of the Rac1 effector PAK1 with a small-molecule inhibitor rescued cofilin signaling in FXS mice, indicating a causal relationship between PAK1 and cofilin signaling. PAK1 inhibition rescued synaptic signaling (specifically the synaptic ratio of NMDA/AMPA in layer V pyramidal neurons) and improved sensory processing in FXS mice. These findings suggest a causal relationship between increased Rac1-cofilin signaling, synaptic defects, and impaired sensory processing in FXS and uncover a previously unappreciated role for impaired Rac1-cofilin signaling in the aberrant spine morphology and spine density associated with FXS.