Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1*

Background: Pathways involved in neuron-dependent GLT1 regulation in astrocytes remain to be characterized. Results: Neuronal microRNA 124a can be transferred into astrocytes through neuronal exosomes and significantly increases GLT1 protein expression in an indirect manner. Conclusion: Neuronal exosomal miRNA 124a is able to regulate astroglial GLT1 expression. Significance: We characterize a novel pathway in neuron-to-astrocyte communication and identify a microRNA that modulates GLT1 protein expression. Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.

[1]  M. Wood,et al.  Exosomes and the emerging field of exosome-based gene therapy. , 2012, Current gene therapy.

[2]  R. Setterquist,et al.  Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. , 2012, Biochimica et biophysica acta.

[3]  E. Bieberich,et al.  Astrocytes Secrete Exosomes Enriched with Proapoptotic Ceramide and Prostate Apoptosis Response 4 (PAR-4) , 2012, The Journal of Biological Chemistry.

[4]  H. Hilton,et al.  The multifaceted exosome: Biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities , 2011, Biochemical Pharmacology.

[5]  C. Théry,et al.  Exosome Secretion: Molecular Mechanisms and Roles in Immune Responses , 2011, Traffic.

[6]  I. Módy,et al.  Deletion of Astroglial Dicer Causes Non-Cell-Autonomous Neuronal Dysfunction and Degeneration , 2011, The Journal of Neuroscience.

[7]  M. Record,et al.  Exosomes as intercellular signalosomes and pharmacological effectors. , 2011, Biochemical pharmacology.

[8]  Ben A. Barres,et al.  Regulation of synaptic connectivity by glia , 2010, Nature.

[9]  Stefano Piccolo,et al.  MicroRNA control of signal transduction , 2010, Nature Reviews Molecular Cell Biology.

[10]  Bulent Ataman,et al.  Trans-Synaptic Transmission of Vesicular Wnt Signals through Evi/Wntless , 2009, Cell.

[11]  C. Théry,et al.  Membrane vesicles as conveyors of immune responses , 2009, Nature Reviews Immunology.

[12]  Lei Wang,et al.  Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport , 2009, Proceedings of the National Academy of Sciences.

[13]  M. Labouesse,et al.  Secretion of Hedgehog‐Related Peptides and WNT During Caenorhabditis elegans Development , 2009, Traffic.

[14]  M. Rattray,et al.  The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies , 2009, Molecular Pharmacology.

[15]  Massimo Spada,et al.  High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients , 2009, PloS one.

[16]  Todd Riley,et al.  The regulation of the endosomal compartment by p53 the tumor suppressor gene , 2009, The FEBS journal.

[17]  Noo Li Jeon,et al.  Presynaptic Regulation of Astroglial Excitatory Neurotransmitter Transporter GLT1 , 2009, Neuron.

[18]  M. Belting,et al.  Nanotubes, exosomes, and nucleic acid–binding peptides provide novel mechanisms of intercellular communication in eukaryotic cells: implications in health and disease , 2008, The Journal of cell biology.

[19]  J. Rothstein,et al.  Focal Transplantation-based Astrocyte Replacement is Neuroprotective in a Model of Motor Neuron Disease , 2008, Nature Neuroscience.

[20]  A. Silahtaroglu,et al.  MicroRNA expression in the adult mouse central nervous system. , 2008, RNA.

[21]  Y. Xing,et al.  A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function , 2008, The Journal of Neuroscience.

[22]  H. Valadi,et al.  Cell to Cell Signalling via Exosomes Through esRNA , 2007, Cell adhesion & migration.

[23]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[24]  Hynek Wichterle,et al.  Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons , 2007, Nature Neuroscience.

[25]  Kevin Eggan,et al.  Non–cell autonomous effect of glia on motor neurons in an embryonic stem cell–based ALS model , 2007, Nature Neuroscience.

[26]  Michael M. Halassa,et al.  The tripartite synapse: roles for gliotransmission in health and disease. , 2007, Trends in molecular medicine.

[27]  Yuan-Fong Lin,et al.  Translational Control of Glial Glutamate Transporter EAAT2 Expression* , 2007, Journal of Biological Chemistry.

[28]  Sascha Keller,et al.  Exosomes: from biogenesis and secretion to biological function. , 2006, Immunology letters.

[29]  D. Turner,et al.  Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[30]  Zuoshang Xu,et al.  Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo , 2006, Neurobiology of Disease.

[31]  G. Lachenal,et al.  Exosomes are released by cultured cortical neurones , 2006, Molecular and Cellular Neuroscience.

[32]  Gail Mandel,et al.  Reciprocal actions of REST and a microRNA promote neuronal identity , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[33]  P. Rosenberg,et al.  Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization , 2005, The Journal of comparative neurology.

[34]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[35]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[36]  C. Aoki,et al.  The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons , 2004, The Journal of Neuroscience.

[37]  D. Volsky,et al.  Cloning and characterization of the 3′‐untranslated region of the human excitatory amino acid transporter 2 transcript , 2003, Journal of neurochemistry.

[38]  Rachel Blitzblau,et al.  Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain , 2002, The Journal of Neuroscience.

[39]  J. Rothstein,et al.  Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS) , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  N. Danbolt.  Glutamate uptake , 2001, Progress in Neurobiology.

[41]  J. Powell,et al.  Intron 7 retention and exon 9 skipping EAAT2 mRNA variants are not associated with amyotrophic lateral sclerosis , 2001, Annals of neurology.

[42]  Lin Jin,et al.  Aberrant RNA Processing in a Neurodegenerative Disease: the Cause for Absent EAAT2, a Glutamate Transporter, in Amyotrophic Lateral Sclerosis , 1998, Neuron.

[43]  J. Rothstein,et al.  Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. , 1998, Molecular pharmacology.

[44]  A. Schousboe,et al.  Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia , 1997, Journal of neurochemistry.

[45]  Y. Kanai,et al.  Tissue specific variants of glutamate transporter GLT‐1 , 1997, FEBS letters.

[46]  J. Rothstein,et al.  Neuronal Regulation of Glutamate Transporter Subtype Expression in Astrocytes , 1997, The Journal of Neuroscience.

[47]  Jeffrey D. Rothstein,et al.  Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex , 1996, Annals of neurology.

[48]  A. Levey,et al.  Selective loss of glial glutamate transporter GLT‐1 in amyotrophic lateral sclerosis , 1995, Annals of neurology.

[49]  A. Levey,et al.  Localization of neuronal and glial glutamate transporters , 1994, Neuron.

[50]  M. Hunter-Ensor,et al.  Pharmacologically distinct sodium-dependentl-[3H]glutamate transport processes in rat brain , 1991, Brain Research.

[51]  A. van Hoof,et al.  Functions of the cytoplasmic exosome. , 2011, Advances in experimental medicine and biology.

[52]  Michael M. Halassa,et al.  The tripartite synapse , 2009 .

[53]  K. Giese,et al.  Expression and Function of Brain Specific Small RNAs , 2004, Reviews in the neurosciences.