Laurie C. Doering

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.

Developmental expression of FMRP in the astrocyte lineage: implications for fragile X syndrome.

One of the most common causes of mental retardation in humans, Fragile X syndrome, results from the absence of FMRP, the protein product of the FMR1 gene. In the nervous system, expression of FMRP has been thought to be confined mainly to neurons as little research has examined FMRP expression in non‐neuronal lineages. We present evidence that, in addition to neuronal expression, FMRP is expressed in developing CNS glial cells in vitro and in vivo. The neurosphere assay was used to establish cultures of stem and progenitor cells from the brains of wildtype and FMRP knockout (B6.129.FMR1/FvBn) mouse pups. When the neurospheres were differentiated in vitro, ∼50% of the FMRP positive cells also expressed GFAP. Immunocytochemical studies of the embryonic and postnatal mouse brain revealed coexpression of FMRP and GFAP in the developing hippocampus. Prominent coexpression was also observed in ependymal cells surrounding the third ventricle and astrocytes of the glia limitans. No double‐labeled cells were evident in the brains of young adult mice. Cells coexpressing FMRP and the oligodendrocyte precursor marker NG2 were also identified in the hippocampus and corpus callosum of the early postnatal brain. Our results suggest that FMRP is expressed in cells of non‐neuronal lineage(s) during development. This represents potential involvement of glial cells in the neural development of fragile X syndrome.

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

BackgroundFragile 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.ResultsWe 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.ConclusionsThese 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.

Reversing autism by targeting downstream mTOR signaling.

Autism spectrum disorders (ASDs) are a group of clinically and genetically heterogeneous neurodevelopmental disorders characterized by impaired social interactions, repetitive behaviors and restricted interests (Baird et al., 2006; Zoghbi and Bear, 2012). The genetic defects in ASDs may interfere with synaptic protein synthesis. Synaptic dysfunction caused by aberrant protein synthesis is a key pathogenic mechanism for ASDs (Kelleher and Bear, 2008; Richter and Klann, 2009; Ebert and Greenberg, 2013). Understanding the details about aberrant synaptic protein synthesis is important to formulate potential treatment for ASDs. The mammalian target of the rapamycin (mTOR) pathway plays central roles in synaptic protein synthesis (Hay and Sonenberg, 2004; Hoeffer and Klann, 2010; Hershey et al., 2012). Recently, Gkogkas and colleagues published exciting data on the role of downstream mTOR pathway in autism (Gkogkas et al., 2013) (Figure u200b(Figure11).

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.

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.

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.

Protocols for neural cell culture

1. Neurosphere and Neural Colony Forming Cell Assays Sharon A. Louis and Brent A. Reynolds 2. Directed Neuronal Differentiation of Embryonic and Adult Derived Neurosphere Cells Marcos R. Costa, Ravi Jagasia, and Benedikt Berninger 3. Culture and Differentiation of Human Neural Stem Cells Soojung Shin and Mohan Vemuri 4. Neural Differentiation of Human Embryonic Stem Cells Mirella Dottori, Alice Pebay, and Martin F. Pera 5. Isolation and Culture of Primary Human CNS Neural Cells Manon Blain, Veronique E. Miron, Caroline Lambert, Peter J. Darlington, Qiao-Ling Cui, Philippe Saikali, V. Wee Yong, and Jack P. Antel 6. Bioengineering Protocols for Neural Precursor Cell Expansion Behnam A. Baghbaderani, Arindom Sen, Michael S. Kallos, and Leo A. Behie 7. Intracellular Calcium Assays in Dissociated Primary Cortical Neurons Navjot Kaur, David V.Thompson, David Judd, David R. Piper, and Richard G. Del Mastro 8. Dissociated Hippocampal Cultures Francine Nault and Paul De Koninck 9. Primary Sensory and Motor Neuron Cultures Andrea M. Vincent and Eva L. Feldman 10. Retinal Cell and Tissue Culture Francisco L.A.F. Gomes and Michel Cayouette 11. Preparation of Normal and Reactive Astrocyte Cultures Jean de Vellis, Cristina A Ghiani, Ina B. Wanner, and Ruth Cole 12. Oligodendrocyte Progenitor Cell Culture Akiko Nishiyama, Ryusuke Suzuki, Hao Zuo, and Xiaoqin Zhu 13. Isolation of Microglia Subpopulations Makoto Sawada and Hiromi Suzuki 14. Microglia from Progenitor Cells in Mouse Neopallium Sergey Fedoroff and Arleen Richardson 15. Primary Schwann Cell Cultures Haesun A. Kim and Patrice Maurel 16. PrimaryDissociated Astrocyte and Neuron Co-Culture Shelley Jacobs and Laurie C. Doering 17. Cerebellar Slice Cultures Josef P. Kapfhammer 18. Hippocampal Slice Cultures Jesse E. Hanson, Adrienne L. Orr, Silvia Fernandez-Illescas, Ricardo A. Valenzuela, and Daniel V. Madison 19. Molecular Substrates for Growing Neurons in Culture Saulius Satkauskas, Arnaud Muller, Morgane Roth, and Dominique Bagnard 20. Guidance and Outgrowth Assays for Embryonic Thalamic Axons Alexandre Bonnin 21. Detection of Cell Death in Neuronal Cultures Sean P. Cregan 22. Live Imaging of Neural Cell Functions Sabine Bavamian, Eliana Scemes, and Paolo Meda 23. Tissue Culture Procedures and Tips Arleen Richardson and Sergey Fedoroff

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

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 99mTc-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 99mTc to visualize these cells in the brain with SPECT. The cells were initially labeled with a permeation peptide carrying a chelate for 99mTc. 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 99mTc-based probe can decrease the cellular proliferation of neural progenitor cells.

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.