Targeting green fluorescent protein to dendritic membrane in central neurons

Dendritic and axonal processes are input and output sites, respectively, of neuronal information, and detailed visualization of these processes may be indispensable for elucidating the neuronal circuits and revealing the principles of neuronal functions. To establish a method for completely visualizing dendritic processes, we first developed green fluorescent protein (GFP)-based proteins and, by using lentivirus with a neuron-specific promoter, examined whether or not the protein fully visualized the dendritic processes of infected neurons. When GFP with a palmitoylation (palGFP) or myristoylation/palmitoylation site (myrGFP) was expressed in rat brain with lentiviruses, myrGFP labeled dendritic membrane better than palGFP. Subsequently, dendrite-targeting efficiencies of three basolateral membrane-sorting and three putative dendrite-targeting domains, which were attached to myrGFP C-terminus, were examined in striatonigral GABAergic and corticothalamic glutamatergic neurons, and in cultured cortical neurons. Of the six domains, C-terminal cytoplasmic domain of low density lipoprotein receptor (LDLRct) was most efficient in targeting the protein to dendrites, showing 8.5-15-fold higher efficiency in striatonigral neurons compared with myrGFP. Finally, dendritic membrane-targeting potency of myrGFP-LDLRct was confirmed in transgenic mice using Thy1 or Gad1 expression cassette. Thus, myrGFP-LDLRct is an excellent synthetic protein for dendritic visualization, and may be a useful tool for the morphological analysis of neuronal circuits.

[1]  T. Murphy,et al.  Regulation of dendritic branching and filopodia formation in hippocampal neurons by specific acylated protein motifs. , 2004, Molecular biology of the cell.

[2]  T. Kaneko,et al.  Immunohistochemical localization of substance P receptor in the central nervous system of the adult rat , 1994, The Journal of comparative neurology.

[3]  T. Kaneko,et al.  Preprodynorphin‐, preproenkephalin‐, and preprotachykinin‐expressing neurons in the rat neostriatum: An analysis by immunocytochemistry and retrograde tracing , 1997, The Journal of comparative neurology.

[4]  D. O'Leary,et al.  Labeling Neural Cells Using Adenoviral Gene Transfer of Membrane-Targeted GFP , 1996, Neuron.

[5]  M. Marks,et al.  Protein sorting by tyrosine-based signals: adapting to the Ys and wherefores. , 1997, Trends in cell biology.

[6]  M. Resh,et al.  Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association. , 1997, Molecular biology of the cell.

[7]  A. Hendrickson,et al.  Distribution of the calcium-binding proteins parvalbumin and calbindin-D28k in the sensorimotor cortex of the rat , 1991, Neuroscience.

[8]  I. Mellman.  Endocytosis and molecular sorting. , 1996, Annual review of cell and developmental biology.

[9]  I. Mellman,et al.  Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells , 1994, The Journal of cell biology.

[10]  M. Resh.  Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. , 1999, Biochimica et biophysica acta.

[11]  Kozo Kaibuchi,et al.  [Neuronal polarity]. , 2008, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[12]  Karen L. Smith,et al.  Novel Hippocampal Interneuronal Subtypes Identified Using Transgenic Mice That Express Green Fluorescent Protein in GABAergic Interneurons , 2000, The Journal of Neuroscience.

[13]  K. Toyama,et al.  Ablation of Cerebellar Golgi Cells Disrupts Synaptic Integration Involving GABA Inhibition and NMDA Receptor Activation in Motor Coordination , 1998, Cell.

[14]  I. Mellman,et al.  Basolateral sorting of LDL receptor in MDCK cells: The cytoplasmic domain contains two tyrosine-dependent targeting determinants , 1992, Cell.

[15]  C. Bennett-Clarke,et al.  Distribution of somatostatin in the rat brain: Telencephalon and diencephalon , 1980, Brain Research.

[16]  K. Simons,et al.  Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture , 1990, Cell.

[17]  G. Westbrook,et al.  Modulation of calcium currents by a metabotropic glutamate receptor involves fast and slow kinetic components in cultured hippocampal neurons , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[18]  T. Kaneko,et al.  In Vivo Transduction of Central Neurons Using Recombinant Sindbis Virus , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[19]  P. Casey,et al.  Protein lipidation in cell signaling. , 1995, Science.

[20]  K. Simons,et al.  Post-Golgi biosynthetic trafficking. , 1997, Journal of cell science.

[21]  Pico Caroni,et al.  Overexpression of growth-associated proteins in the neurons of adult transgenic mice , 1997, Journal of Neuroscience Methods.

[22]  M. Eiraku,et al.  Delta/Notch-like Epidermal Growth Factor (EGF)-related Receptor, a Novel EGF-like Repeat-containing Protein Targeted to Dendrites of Developing and Adult Central Nervous System Neurons* , 2002, The Journal of Biological Chemistry.

[23]  M. Resh,et al.  Rapid Plasma Membrane Anchoring of Newly Synthesized p59  fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3 , 1997, The Journal of cell biology.

[24]  T. Hope,et al.  Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors , 1999, Journal of Virology.

[25]  KouichiC . Nakamura,et al.  Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters , 2007, Gene Therapy.

[26]  T. Kaneko,et al.  Predominant information transfer from layer III pyramidal neurons to corticospinal neurons , 2000, The Journal of comparative neurology.

[27]  K. Mori,et al.  A Novel Phenylalanine-Based Targeting Signal Directs Telencephalin to Neuronal Dendrites , 2005, The Journal of Neuroscience.

[28]  E G Jones,et al.  Visualization of chandelier cell axons by parvalbumin immunoreactivity in monkey cerebral cortex. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Takeshi Kaneko,et al.  Intracellularly labeled pyramidal neurons in the cortical areas projecting to the spinal cord II. Intra- and juxta-columnar projection of pyramidal neurons to corticospinal neurons , 2004, Neuroscience Research.

[30]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[31]  I. Katona,et al.  In Vivo Labeling of Parvalbumin-Positive Interneurons and Analysis of Electrical Coupling in Identified Neurons , 2002, The Journal of Neuroscience.

[32]  KouichiC . Nakamura,et al.  Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo , 2000, Neuroscience Research.

[33]  T. Kaneko,et al.  Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67‐GFP knock‐in mouse , 2003, The Journal of comparative neurology.

[34]  G. Banker,et al.  The Polarized Sorting of Membrane Proteins Expressed in Cultured Hippocampal Neurons Using Viral Vectors , 1998, Neuron.