Optimization of a 96-Well Electroporation Assay for Postnatal Rat CNS Neurons Suitable for Cost–Effective Medium-Throughput Screening of Genes that Promote Neurite Outgrowth

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

[1]  J Teissié,et al.  Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane. , 1997, Biophysical journal.

[2]  E Neumann,et al.  Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. , 1994, Biophysical journal.

[3]  Edgar Erdfelder,et al.  G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences , 2007, Behavior research methods.

[4]  D. Snow,et al.  Binding characteristics of chondroitin sulfate proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard tissue culture choice assay. , 2002, Journal of Neurobiology.

[5]  M. Kiebler,et al.  High-efficiency transfection of mammalian neurons via nucleofection , 2007, Nature Protocols.

[6]  K. Schilling,et al.  Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro , 2002, Histochemistry and Cell Biology.

[7]  Robert Nadon,et al.  Statistical practice in high-throughput screening data analysis , 2006, Nature Biotechnology.

[8]  Helmut Mack,et al.  Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo , 2007, Journal of neurochemistry.

[9]  R. Meldrum,et al.  Optimisation of electroporation for biochemical experiments in live cells. , 1999, Biochemical and biophysical research communications.

[10]  M. Ruonala,et al.  Rapid and efficient electroporation-based gene transfer into primary dissociated neurons , 2003, Journal of Neuroscience Methods.

[11]  C. Bandtlow,et al.  Nogo-A and Myelin-Associated Glycoprotein Mediate Neurite Growth Inhibition by Antagonistic Regulation of RhoA and Rac1 , 2002, The Journal of Neuroscience.

[12]  J. Teissié,et al.  Generation of reactive-oxygen species induced by electropermeabilization of Chinese hamster ovary cells and their consequence on cell viability. , 1994, European journal of biochemistry.

[13]  M. Berry,et al.  Effective gene delivery to adult neurons by a modified form of electroporation , 2005, Journal of Neuroscience Methods.

[14]  J. Silver,et al.  Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro , 1990, Experimental Neurology.

[15]  M. Filbin,et al.  A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration , 1994, Neuron.

[16]  Vance P Lemmon,et al.  Kinase/phosphatase overexpression reveals pathways regulating hippocampal neuron morphology , 2010, Molecular systems biology.

[17]  L. Collin,et al.  Nucleofection of primary neurons. , 2006, Methods in enzymology.

[18]  Zhigang He,et al.  PKC mediates inhibitory effects of myelin and chondroitin sulfate proteoglycans on axonal regeneration , 2004, Nature Neuroscience.

[19]  M. Rols,et al.  Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge. , 2005, Biochimica et biophysica acta.

[20]  K. Kaestner,et al.  KLF Family Members Regulate Intrinsic Axon Regeneration Ability , 2009, Science.

[21]  J Teissié,et al.  Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. , 1998, Biophysical journal.

[22]  Marco Domeniconi,et al.  MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth , 2005, Neuron.

[23]  M. Tessier-Lavigne,et al.  The Nogo-66 Receptor NgR1 Is Required Only for the Acute Growth Cone-Collapsing But Not the Chronic Growth-Inhibitory Actions of Myelin Inhibitors , 2007, The Journal of Neuroscience.

[24]  J. Bixby,et al.  96-well electroporation method for transfection of mammalian central neurons. , 2006, BioTechniques.

[25]  E Neumann,et al.  Fundamentals of electroporative delivery of drugs and genes. , 1999, Bioelectrochemistry and bioenergetics.

[26]  J. Schwab,et al.  The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar , 2003, Molecular and Cellular Neuroscience.

[27]  D. Snow,et al.  Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG). , 1992, Journal of neurobiology.

[28]  R. Smith,et al.  A quantitative method for analysis of in vitro neurite outgrowth , 2007, Journal of Neuroscience Methods.

[29]  M. Filbin,et al.  Myelin-Associated Glycoprotein Inhibits Axonal Regeneration from a Variety of Neurons via Interaction with a Sialoglycoprotein , 1996, Molecular and Cellular Neuroscience.

[30]  M. Rols,et al.  Temperature effects on electrotransfection of mammalian cells. , 1994, Nucleic acids research.

[31]  C. Woolf,et al.  ATF3 Increases the Intrinsic Growth State of DRG Neurons to Enhance Peripheral Nerve Regeneration , 2007, The Journal of Neuroscience.

[32]  J Teissié,et al.  Control by electrical parameters of short- and long-term cell death resulting from electropermeabilization of Chinese hamster ovary cells. , 1995, Biochimica et biophysica acta.

[33]  John L. Bixby,et al.  High content screening of cortical neurons identifies novel regulators of axon growth , 2010, Molecular and Cellular Neuroscience.

[34]  R. Giger,et al.  The Nogo-66 Receptor Homolog NgR2 Is a Sialic Acid-Dependent Receptor Selective for Myelin-Associated Glycoprotein , 2005, The Journal of Neuroscience.

[35]  A. McAllister,et al.  Techniques for gene transfer into neurons , 2002, Current Opinion in Neurobiology.