Technical Reference #1699

1699.

Importin-Mediated Retrograde Transport of CREB2 From Distal Processes to the Nucleus in Neurons Kwok-On Lai; Yali Zhaoa;b; Toh Hean Ch'nga;b; and Kelsey C. Martin, University of California, PNAS, 105(1699), (2008)
Link To Paper

Abstract:
Signals received at distal synapses of neurons must be conveyed to the nucleus to initiate the changes in transcription that underlie long-lasting synaptic plasticity. The presence of importin nuclear transporters and of select transcription factors at synapses raises the possibility that importins directly transport transcription factors from synapse to nucleus to modulate gene expression. Here we show that cyclic AMP response element binding protein 2 (CREB2)/activating transcription factor 4 (ATF4) a transcriptional repressor that modulates long-term synaptic plasticity and memory localizes to distal dendrites of rodent hippocampal neurons and neurites of Aplysia sensory neurons (SNs) and binds to specific importin isoforms. Binding of CREB2 to importin is required for its transport from distal dendrites to the soma and for its translocation into the nucleus. CREB2 accumulates in the nucleus during long-term depression (LTD) but not long-term potentiation of rodent hippocampal synapses and during LTD but not long-term facilitation (LTF) of Aplysia sensory-motor synapses. Time-lapse microscopy of CREB2 tagged with a photoconvertible fluorescent protein further reveals retrograde transport of CREB2 from distal neurites to the nucleus of Aplysia SN during phenylalaninemethionine- arginine-phenylalanine-amide (FMRFamide)-induced LTD. Together our findings indicate that CREB2 is a novel cargo of importin that translocates from distal synaptic sites to the nucleus after stimuli that induce LTD of neuronal synapses.

Keywords:
aplysia; hippocampal; transcription; long-term depression; dendra

Materials & Methods:
Immunofluorescence Staining and in Situ Hybridization. Aplysia neurons were fixed in ice-cold 4% paraformaldehyde/30% sucrose for 15min at room temp. After washing three times with PBS containing 30% sucrose cells were permeabilized with 0.1% Triton-X in PBS/30% sucrose for 5 min washed three times with PBS and quenched with 50 mM ammonium chloride for 15 min. The cells were then washed in PBS three times and blocked with PBS containing10% normal goat serum for 1.5 h before incubation with primary antibodies (diluted in PBS/10% normal goat serum) overnight at 4 °C. Cells were then washed five times in PBS and incubated with Alexa Fluor conjugated secondary antibodies (Invitrogen/Molecular Probes) for 1 h at room temp. After washing six times with PBS the cells were imaged by laser-scanning confocal microscopy. The procedures for hippocampal neurons and 293T cells were essentially the same except that sucrose was omitted from the fixative and permeabilizing solution.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
Neurons - Hippocampal, 293T