Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures

Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedited technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova etal., 1997), and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

[1]  Frank W. Pfrieger,et al.  Multiple mechanisms mediate cholesterol-induced synaptogenesis in a CNS neuron , 2005, Molecular and Cellular Neuroscience.

[2]  A. Ferreira,et al.  Progesterone-induced agrin expression in astrocytes modulates glia–neuron interactions leading to synapse formation , 2006, Neuroscience.

[3]  C. ffrench-Constant,et al.  In vitro modeling of central nervous system myelination and remyelination , 2012, Glia.

[4]  T. Basarsky,et al.  Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[5]  R. Campenot,et al.  Glial Lipoproteins Stimulate Axon Growth of Central Nervous System Neurons in Compartmented Cultures* , 2004, Journal of Biological Chemistry.

[6]  A. Smit,et al.  SCAP is required for timely and proper myelin membrane synthesis , 2009, Proceedings of the National Academy of Sciences.

[7]  L. Roux,et al.  Over Astroglial Networks: a Step Further in Neuroglial and Gliovascular Interactions , 2022 .

[8]  Thomas Lengauer,et al.  Improved scoring of functional groups from gene expression data by decorrelating GO graph structure , 2006, Bioinform..

[9]  Matthias E. Futschik,et al.  OLIN: optimized normalization, visualization and quality testing of two-channel microarray data , 2005, Bioinform..

[10]  Michael M. Halassa,et al.  Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. , 2010, Annual review of physiology.

[11]  A. Smit,et al.  SREBPs: SREBP function in glia–neuron interactions , 2009, The FEBS journal.

[12]  M. Verhage,et al.  Interdependence of PKC-Dependent and PKC-Independent Pathways for Presynaptic Plasticity , 2007, Neuron.

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

[14]  E. López-Bayghen,et al.  Glutamate regulates Oct‐2 DNA‐binding activity through α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors in cultured chick Bergmann glia cells , 2004, Journal of neurochemistry.

[15]  K. Zou,et al.  Cholesterol‐dependent modulation of dendrite outgrowth 
and microtubule stability in cultured neurons , 2002, Journal of neurochemistry.

[16]  John D. Lambris,et al.  The Classical Complement Cascade Mediates CNS Synapse Elimination , 2007, Cell.

[17]  J. Medina,et al.  Astrocyte-synthesized oleic acid behaves as a neurotrophic factor for neurons , 2002, Journal of Physiology-Paris.

[18]  L. Foo Purification of rat and mouse astrocytes by immunopanning. , 2013, Cold Spring Harbor protocols.

[19]  R. Hammer,et al.  Schoenheimer effect explained--feedback regulation of cholesterol synthesis in mice mediated by Insig proteins. , 2005, The Journal of clinical investigation.

[20]  F. Pfrieger,et al.  Cholesterol metabolism in neurons and astrocytes. , 2011, Progress in lipid research.

[21]  S. Goldman,et al.  The Transcriptome and Metabolic Gene Signature of Protoplasmic Astrocytes in the Adult Murine Cortex , 2007, The Journal of Neuroscience.

[22]  J. Hell,et al.  Thrombospondins Are Astrocyte-Secreted Proteins that Promote CNS Synaptogenesis , 2005, Cell.

[23]  L. Acarín,et al.  Glial activation in the immature rat brain: implication of inflammatory transcription factors and cytokine expression. , 2001, Progress in brain research.

[24]  C. Göritz,et al.  CNS synaptogenesis promoted by glia-derived cholesterol. , 2001, Science.

[25]  Ben A. Barres,et al.  Emerging roles of astrocytes in neural circuit development , 2013, Nature Reviews Neuroscience.

[26]  V. Mandys,et al.  Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment , 1997, Journal of Neuroscience Methods.

[27]  B. Barres,et al.  Control of synapse number by glia. , 2001, Science.