Regulation of IL-6 Secretion by Astrocytes via TLR4 in the Fragile X Mouse Model.

Fragile X syndrome (FXS) is identified by abnormal dendrite morphology and altered synaptic protein expression. Astrocyte secreted factors such as Tenascin C (TNC), may contribute to the synaptic changes, including maturation of the synapse. TNC is a known endogenous ligand of toll-like receptor 4 (TLR4) that has been shown to induce the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). At the molecular level, elevated IL-6 promotes excitatory synapse formation and increases dendrite spine length. With these molecular changes linked to the phenotype of FXS, we examined the expression and the mechanism of the endogenous TLR4 activator TNC, and its downstream target IL-6 in astrocytes from the Fragile X Mental Retardation 1 (FMR1) knockout (KO) mouse model. Secreted TNC and IL-6 were significantly increased in FMR1 KO astrocytes. Addition of TNC and lipopolysaccharide (LPS) induced IL-6 secretion, whereas the antagonist of TLR4 (LPS-RS) had an opposing effect. Cortical protein expression of TNC and IL-6 were also significantly elevated in the postnatal FMR1 KO mouse. In addition, there was an increase in the number of vesicular glutamate transporter 1 (VGLUT1)/post synaptic density protein 95 (PSD95) positive synaptic puncta of both wild-type (WT) and FMR1 KO neurons when plated with astrocyte conditioned media (ACM) from FMR1 KO astrocytes, compared to those plated with media from wild type astrocytes. By assessing the cellular mechanisms involved, a novel therapeutic option could be made available to target abnormalities of synaptic function seen in FXS.

Altered Developmental Expression of the Astrocyte-Secreted Factors Hevin and SPARC in the Fragile X Mouse Model.

Astrocyte dysfunction has been indicated in many neurodevelopmental disorders, including Fragile X Syndrome (FXS). FXS is caused by a deficiency in fragile X mental retardation protein (FMRP). FMRP regulates the translation of numerous mRNAs and its loss disturbs the composition of proteins important for dendritic spine and synapse development. Here, we investigated whether the astrocyte-derived factors hevin and SPARC, known to regulate excitatory synapse development, have altered expression in FXS. Specifically, we analyzed the expression of these factors in wild-type (WT) mice and in fragile X mental retardation 1 (Fmr1) knock-out (KO) mice that lack FMRP expression. Samples were collected from the developing cortex and hippocampus (regions of dendritic spine abnormalities in FXS) of Fmr1 KO and WT pups. Hevin and SPARC showed altered expression patterns in Fmr1 KO mice compared to WT, in a brain-region specific manner. In cortical tissue, we found a transient increase in the level of hevin in postnatal day (P)14 Fmr1 KO mice, compared to WT. Additionally, there were modest decreases in Fmr1 KO cortical levels of SPARC at P7 and P14. In the hippocampus, hevin expression was much lower in P7 Fmr1 KO mice than in WT. At P14, hippocampal hevin levels were similar between genotypes, and by P21 Fmr1 KO hevin expression surpassed WT levels. These findings imply aberrant astrocyte signaling in FXS and suggest that the altered expression of hevin and SPARC contributes to abnormal synaptic development in FXS.

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST+CD15+CD133+) and an increased proportion of neuroblasts (PSA-NCAM+CD15+) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G2/M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X.

Astrocyte-secreted thrombospondin-1 modulates synapse and spine defects in the fragile X mouse model.

Astrocytes are key participants in various aspects of brain development and function, many of which are executed via secreted proteins. Defects in astrocyte signaling are implicated in neurodevelopmental disorders characterized by abnormal neural circuitry such as Fragile X syndrome (FXS). In animal models of FXS, the loss in expression of the Fragile X mental retardation 1 protein (FMRP) from astrocytes is associated with delayed dendrite maturation and improper synapse formation; however, the effect of astrocyte-derived factors on the development of neurons is not known. Thrombospondin-1 (TSP-1) is an important astrocyte-secreted protein that is involved in the regulation of spine development and synaptogenesis. In this study, we found that cultured astrocytes isolated from an Fmr1 knockout (Fmr1 KO) mouse model of FXS displayed a significant decrease in TSP-1 protein expression compared to the wildtype (WT) astrocytes. Correspondingly, Fmr1 KO hippocampal neurons exhibited morphological deficits in dendritic spines and alterations in excitatory synapse formation following long-term culture. All spine and synaptic abnormalities were prevented in the presence of either astrocyte-conditioned media or a feeder layer derived from FMRP-expressing astrocytes, or following the application of exogenous TSP-1. Importantly, this work demonstrates the integral role of astrocyte-secreted signals in the establishment of neuronal communication and identifies soluble TSP-1 as a potential therapeutic target for Fragile X syndrome.

Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse.

UNLABELLED: An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. HIGHLIGHTS: • We studied the proliferation of neural stem and progenitor cells in Fragile X.• We examined the role of astrocyte-secreted factors in neural precursor cell biology.• Astrocyte-secreted factors with differential expression in Fragile X identified.

Developmental expression of the neuroligins and neurexins in fragile X mice.

Neuroligins and neurexins are transsynaptic proteins involved in the maturation of glutamatergic and GABAergic synapses. Research has identified synaptic proteins and function as primary contributors to the development of fragile X syndrome. Fragile X mental retardation protein (FMRP), the protein that is lacking in fragile X syndrome, binds neuroligin-1 and -3 mRNA. Using in situ hybridization, we examined temporal and spatial expression patterns of neuroligin (NLGN) and neurexin (NRXN) mRNAs in the somatosensory (S1) cortex and hippocampus in wild-type (WT) and fragile X knockout (FMR1-KO) mice during the first 5 weeks of postnatal life. Genotype-based differences in expression included increased NLGN1 mRNA in CA1 and S1 cortex, decreased NLGN2 mRNA in CA1 and dentate gyrus (DG) regions of the hippocampus, and increased NRXN3 mRNA in CA1, DG, and S1 cortex between female WT and FMR1-KO mice. In male mice, decreased expression of NRXN3 mRNA was observed in CA1 and DG regions of FMR1-KO mice. Sex differences in hippocampal expression of NLGN2, NRXN1, NRXN2, and NRXN3 mRNAs and in S1 cortex expression of NRXN3 mRNAs were observed WT mice, whereas sex differences in NLGN3, NRXN1, NRXN2, and NRXN3 mRNA expression in the hippocampus and in NLGN1, NRXN2 and NRXN3 mRNA expression in S1 cortex were detected in FMR1-KO mice. These results provide a neuroanatomical map of NLGN and NRXN expression patterns over postnatal development in WT and FMR1-KO mice. The differences in developmental trajectory of these synaptic proteins could contribute to long-term differences in CNS wiring and synaptic function.

Targeted pharmacological treatment of autism spectrum disorders: fragile X and Rett syndromes.

Autism spectrum disorders (ASDs) are genetically and clinically heterogeneous and lack effective medications to treat their core symptoms. Studies of syndromic ASDs caused by single gene mutations have provided insights into the pathophysiology of autism. Fragile X and Rett syndromes belong to the syndromic ASDs in which preclinical studies have identified rational targets for drug therapies focused on correcting underlying neural dysfunction. These preclinical discoveries are increasingly translating into exciting human clinical trials. Since there are significant molecular and neurobiological overlaps among ASDs, targeted treatments developed for fragile X and Rett syndromes may be helpful for autism of different etiologies. Here, we review the targeted pharmacological treatment of fragile X and Rett syndromes and discuss related issues in both preclinical studies and clinical trials of potential therapies for the diseases.

Fluorescent labeling of dendritic spines in cell cultures with the carbocyanine dye "DiI".

Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system.

Temporal and spectral differences in the ultrasonic vocalizations of fragile X knock out mice during postnatal development.

The fmr1 knock out (KO) mouse has been a useful animal model to understand pathology and treatment of FXS, both anatomically and behaviorally. Ultrasonic vocalizations (USVs) are a behavioral tool to assess early life communication deficits in mice. Here, we report on the temporal and spectral features of USVs emitted after maternal separation in wild type (FVB/N) and fmr1 KO pups at postnatal days (P) P4, P7 and P10. The results show changes in the number and duration of calls in fmr1 KO pups and wild type pups were dependent on age and call type. Fmr1 KO pups showed an increased number of USVs at P7 but not at P4 or P10. This increase was specific to Frequency Jump calls. In addition, fmr1 KO mice showed a developmental shift in the temporal distribution of calls, with P10 mice calling in distinct bout patterns. Overall, these findings provide evidence that changes in USV outcomes were specific to certain call types and ages in fmr1 KO mice. Because early postnatal life is a window during which multiple neural systems activate and become established, behavioral measures such as using USVs as a measure of communication, may be useful as a predictor of brain changes and later developmental behavioral changes. Work is needed to better understand the functional outcomes of altered development of USVs and how these changes contribute to later emergence of autistic-like behaviors in animal models of autism.

Progress toward therapeutic potential for AFQ056 in Fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading single-gene cause of autism. It is caused by the lack of production of the Fragile X mental retardation protein (FMRP), resulting in cognitive deficits, hyperactivity, and autistic behaviors. Breakthrough advances in potential therapy for FXS followed the discovery that aberrant group 1 metabotropic glutamate receptor (mGluR) signaling is an important constituent of the pathophysiology of the syndrome. Research has indicated that upon neuronal stimulation, FMRP acts downstream of group 1 mGluRs (mGluRs1/5) to inhibit protein synthesis, long-term depression, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor internalization. To offset the deficits caused by the lack of FMRP, many pharmaceutical companies have designed medicinal drugs to target the unrestrained stimulation of mGluR5 signaling in FXS. Indeed, promising results from animal and clinical studies suggest that mGluR5 antagonists such as AFQ056 can successfully correct many of the deficits in FXS. In this review, we cover the animal studies performed to date that test the role of AFQ056 as a selective mGluR5 antagonist to alleviate the phenotypes of FXS.

Induced pluripotent stem cells to model and treat neurogenetic disorders.

Remarkable advances in cellular reprogramming have made it possible to generate pluripotent stem cells from somatic cells, such as fibroblasts obtained from human skin biopsies. As a result, human diseases can now be investigated in relevant cell populations derived from induced pluripotent stem cells (iPSCs) of patients. The rapid growth of iPSC technology has turned these cells into multipurpose basic and clinical research tools. In this paper, we highlight the roles of iPSC technology that are helping us to understand and potentially treat neurological diseases. Recent studies using iPSCs to model various neurogenetic disorders are summarized, and we discuss the therapeutic implications of iPSCs, including drug screening and cell therapy for neurogenetic disorders. Although iPSCs have been used in animal models with promising results to treat neurogenetic disorders, there are still many issues associated with reprogramming that must be addressed before iPSC technology can be fully exploited with translation to the clinic.

Astrocytes and developmental plasticity in fragile X.

A growing body of research indicates a pivotal role for astrocytes at the developing synapse. In particular, astrocytes are dynamically involved in governing synapse structure, function, and plasticity. In the postnatal brain, their appearance at synapses coincides with periods of developmental plasticity when neural circuits are refined and established. Alterations in the partnership between astrocytes and neurons have now emerged as important mechanisms that underlie neuropathology. With overall synaptic function standing as a prominent link to the expression of the disease phenotype in a number of neurodevelopmental disorders and knowing that astrocytes influence synapse development and function, this paper highlights the current knowledge of astrocyte biology with a focus on their involvement in fragile X syndrome.

Probing astrocyte function in fragile X syndrome.

Astrocytes have been recognized as a class of cells that fill the space between neurons for more than a century. From their humble beginnings in the literature as merely space filling cells, an ever expanding list of functions in the CNS now exceeds the list of functions performed by neurons. In virtually all developmental and pathological conditions in the brain, astrocytes are involved in some capacity that directly affects neuronal function. Today we recognize that astrocytes are involved in the development and function of synaptic communication. Increasing evidence suggests that abnormal synaptic function may be a prominent contributing factor to the learning disability phenotype. With the discovery of FMRP in astrocytes, coupled with a role of astrocytes in synaptic function, research directed to glial neurobiology has never been more important. This chapter highlights the current knowledge of astrocyte function with a focus on their involvement in Fragile X syndrome.

Correlative single photon emission computed tomography imaging of [123I]altropane binding in the rat model of Parkinson's.

INTRODUCTION: This study used the dopamine transporter (DAT) probe, [(123)I]-2ß-carbomethoxy-3ß-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane ([(123)I]altropane), to assess the DAT levels in the 6-hydroxydopamine rat model of Parkinson's disease. We sought to assess if the right to left [(123)I]altropane striatal ratios correlated with dopamine content in the striatum and substantia nigra and with behavioural outcomes. METHODS: [(123)I]altropane images taken pre- and postlesion were acquired before and after the transplantation of neural stem/progenitor cells. The images obtained using [(123)I]altropane and single photon emission computed tomography (SPECT) were compared with specific behavioural tests and the dopamine content assessed by high-performance liquid chromatography. RESULTS: [(123)I]altropane binding correlated with the content of dopamine in the striatum; however, [(123)I]altropane binding did not correlate with the dopamine content in the substantia nigra. There was a significant correlation of altropane ratios with the cylinder test and the postural instability test, but not with amphetamine rotations. The low coefficient of determination (r(2)) for these correlations indicated that [(123)I]altropane SPECT was not a good predictor of behavioural outcomes. CONCLUSION: Our data reveal that [(123)I]altropane predicts the integrity of the striatal dopamine nerve terminals, but does not predict the integrity of the nigrostriatal system. [(123)I]altropane could be a useful marker to measure dopamine content in cell replacement therapies; however, it would not be able to evaluate outcomes for neuroprotective strategies.

99mTc-based imaging of transplanted neural stem cells and progenitor cells.

UNLABELLED: Cell therapy for neurologic disorders will benefit significantly from progress in methods of noninvasively imaging cell transplants. The success of current cell therapy has varied, in part because of differences in cell sources, differences in transplantation procedures, and lack of understanding of cell fate after transplantation. Standardization of transplantation procedures will progress with noninvasive imaging. In turn, in vivo imaging will enhance our understanding of neural transplant biology and improve therapeutic outcomes. The goal of this study was to determine the effect of a (99m)Tc-based probe on neural stem and progenitor cell transplants and validate the SPECT images of the transplanted cells. METHODS: We previously developed a method to label neural stem and progenitor cells with (99m)Tc to visualize these cells in the brain with SPECT. The cells were initially labeled with a permeation peptide carrying a chelate for (99m)Tc. The proliferation and differentiation characteristics of the labeled cells were studied in tissue culture. In parallel experiments, the labeled cells were stereotactically injected into the rat brain, and the site of transplantation was verified with histochemistry and phosphorimaging. RESULTS: The accuracy of the transplant location obtained by SPECT was confirmed by comparison with phosphorimages and histologic sections of the brain. The labeling did, however, decrease the proliferative capacity of the neural stem and progenitor cells. CONCLUSION: The labeling technique described here can be used to standardize the location of cell transplants in the brain and quantify the number of transplanted cells. However, a (99m)Tc-based probe can decrease the cellular proliferation of neural progenitor cells.

Fragile X astrocytes induce developmental delays in dendrite maturation and synaptic protein expression.

BACKGROUND: Fragile X syndrome is the most common inherited form of mental impairment characterized by cognitive impairment, attention deficit and autistic behaviours. The mouse model of Fragile X is used to study the underlying neurobiology associated with behavioral deficiencies. The effect of Fragile X glial cells on the development of neurons has not been studied. We used a co-culture technique in combination with morphometrics on immunostained neurons to investigate the role of astrocytes in the development delays associated with hippocampal neuron development. RESULTS: We found that hippocampal neurons grown on Fragile X astrocytes exhibited a significant difference from the neurons grown with normal astrocytes after 7 days in vitro for many parameters including increases in dendritic branching and in area of the cell body. However, after 21 days in culture, the neurons grown on Fragile X astrocytes exhibited morphological characteristics that did not differ significantly from the neurons grown on normal astrocytes. With antibodies to the pre-synaptic protein, synapsin, and to the excitatory post-synaptic protein, PSD-95, we quantified the number of developing excitatory synapses on the dendrites. In addition to the delays in dendritic patterning, the development of excitatory synapses was also delayed in the hippocampal neurons. CONCLUSIONS: These experiments are the first to establish a role for astrocytes in the delayed growth characteristics and abnormal morphological features in dendrites and synapses that characterize the Fragile X syndrome.

Astrocytes prevent abnormal neuronal development in the fragile x mouse.

Astrocytes are now distinguished as major regulators of neuronal growth and synaptic development. Recently, they have been identified as key players in the progression of a number of developmental disorders; however, in fragile X syndrome (FXS), the role of astrocytes is not known. Using a coculture design, we found that hippocampal neurons exhibited abnormal dendritic morphology and a decreased number of presynaptic and postsynaptic protein aggregates when they were grown on astrocytes from a fragile X mouse. Moreover, we found that normal astrocytes could prevent the development of abnormal dendrite morphology and preclude the reduction of presynaptic and postsynaptic protein clusters in neurons from a fragile X mouse. These experiments are the first to establish a role for astrocytes in the altered neurobiology of FXS. Our results support the notion that astrocytes contribute to abnormal dendrite morphology and the dysregulated synapse development in FXS.