A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation - PubMed (original) (raw)

A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation

M Matsushita et al. J Neurosci. 2001.

Abstract

Proteins and peptides have been demonstrated to penetrate across the plasma membrane of eukaryotic cells by protein transduction domains. We show that protein transduction by 11 arginine (11R) is an efficient method of delivering proteins into the neurons of brain slices. Here, we demonstrate that PKA inhibitory peptide, fused with 11R and nuclear localization signal, delivers the peptide exclusively into the nuclear compartment of neurons in brain slices. This inhibitory peptide blocked both cAMP responsive element-binding protein phosphorylation and long-lasting long-term potentiation (LTP) induction, but not early LTP. These results highlight transduction of proteins and peptides into specific neuronal subcellular compartments in brain slices as a powerful tool for studying neuronal plasticity.

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Figures

Fig. 1.

Fig. 1.

Transduction of a series of PTD-EGFP proteins into Cos-7 cells. A, Schematic representation of EGFP and PTD-EGFPs. A series of DNAs were amplified using PCR from cDNA encoding the EGFP (4). B, Analysis of protein transduction in Cos-7 cells by confocal microscopy. Cos-7 cells cultured on glass coverslips were incubated with 1 μ

m

EGFP, TAT-EGFP,_11R_-EGFP, _9R_-EGFP, and_7R_-EGFP. After 30 min, cells were washed three times and incubated another 30 min and then analyzed by confocal microscopy. Laser power was identical within each experiment. Scale bar, 100 μm. C, The intracellular presence of the EGFP was analyzed by Western blotting using GFP monoclonal antibody; then, signals were analyzed by NIH Image (n = 3).

Fig. 2.

Fig. 2.

Cell type specificity of _11R_protein transduction domain. A, _11R_-EGFP protein (1 μ

m)

was incubated with cells for 30 min, after which the medium was changed, incubated for another 30 min, and washed three times with PBS. The cells were analyzed by confocal microscopy. Laser power was identical within each experiment. PC12 cells were treated with 100 ng/ml NGF (Life Technologies) in DMEM (Life Technologies) with 0.5% FBS for 2 d. The intracellular presence of the _11R_-EGFP (B) and TAT-EGFP (C) in PC12 cells was analyzed by Western blotting using GFP monoclonal antibody; then, signals were analyzed by NIH Image (n = 3). The data shown are the average of three experiments. Three independent experiments gave similar results.

Fig. 3.

Fig. 3.

Transduction of _11R_-EGFP into a hippocampal brain slice. Protein (1 μ

m)

was incubated with the brain slice for 30 min, after which the medium was changed, incubated for another 30 min, and washed three times with PBS. The slices were analyzed by confocal microscopy. _11R_-EGFP was transduced in the neurons of hippocampus. EGFP signals in neurons were examined using a Zeiss confocal microscope._11R_-EGFP was transduced into the neurons in the CA1 region (A), and CA4 and dentate gyrus (DG) (B). Scale bars, 100 μm.C, The images are multiple optical 12 μm step sections spanning the _Z-_dimension of laser scans of the area CA2–CA3 of hippocampal slice.

Fig. 4.

Fig. 4.

Distribution of _11R_-EGFP in a hippocampal brain slice. A_–_C, Double staining with EGFP (green) and rhodamine-conjugated second antibody against synapsin antibody (red). D_–_F, Double staining with EGFP (green) and rhodamine-conjugated second antibody against GFAP antibody (red). MF, Mossy fiber;DG, dentate gyrus. Scale bars:A_–_C, 50 μm;D_–_F, 200 μm.

Fig. 5.

Fig. 5.

Analysis of subcellular localization of NLS-_11R_-EGFP, FITC-_11R_-PKI, and FITC-NLS-_11R_-PKI in a hippocampal slice and primary culture neurons. A, Transduction of NLS-_11R_-EGFP into a hippocampal brain slice. Protein (1 μ

m)

was incubated with the brain slice for 30 min, after which the medium was changed and incubated for another 30 min. The CA1 region of slices was analyzed by confocal microscopy. B, Schematic representation of synthesized PKA inhibitor peptides.C, Subcellular localization of FITC-_11R_-PKI and FITC-NLS-_11R_-PKI in 10 d primary culture neurons. Primary culture neurons were incubated with each peptide (1 μ

m)

for 30 min, after which the medium was changed and incubated for another 30 min. Culture cells were fixed by 4% paraformaldehyde and then analyzed by confocal microscopy. Scale bars, 100 μm.

Fig. 6.

Fig. 6.

Inhibition of PKA activity in brain slices.A, Transduction of FITC-NLS-_11R_-PKI into a hippocampal brain slice. Peptide (1 μ

m)

was incubated with the brain slice for 30 min, after which the medium was changed, incubated for another 30 min, washed three times with PBS, and then fixed with 4% paraformaldehyde. The fixed slices were stained with MAP-2 monoclonal antibody. FITC signals in neurons of the brain slices were examined using a Zeiss confocal microscope. FITC-NLS-_11R_-PKI was localized in the nucleus. Pyramidal neurons in CA1 were detected by MAP-2 antibody (red). The nuclei of pyramidal neurons showed the green FITC-NLS-_11R_-PKI signal. Scale bar, 50 μm.B, Reduced phosphorylation of CREB at serine 133 by_11R_-PKI and NLS-_11R_-PKI. Hippocampal brain slices were incubated at the indicated peptide concentration and control (absence of peptide) for 1 hr, after which the ACSF was changed and incubated 30 min. The slices were stimulated by 10 μ

m

of forskolin (Forsk) for 10 min. The reactions were immediately stopped by adding 0.1% SDS into the slices. Then, samples were analyzed by Western blotting using phospho-Ser133 CREB specific antibody, phospho-Ser 845 GluR1 antibody, and actin antibody (n = 3). C,_11R_-PKI did not inhibit the MAP kinase cascade. Hippocampal brain slices were incubated at the indicated peptide concentration, control (Cont; absence of peptide), and 10 μ

m

U0126 for 30 min, after which the ACSF was changed and incubated 30 min. The slices were stimulated by 100 μ

m

glutamate (Glu) for 10 min. Samples were analyzed by Western blotting using phospho-p42/44 MAP kinase antibody and actin antibody (n = 3). Three independent experiments gave similar results.

Fig. 7.

Fig. 7.

NLS-_11R_-PKI peptides inhibited the L-LTP induction, but not that of E-LTP. A, NLS-_11R_-PKI did not block E-LTP induced by one train of 100 Hz for 1 sec tetanization (arrow). B, NLS-_11R_-PKI significantly inhibited L-LTP induced by three trains of 100 Hz for 1 sec tetanization (arrows). In contrast, NLS-_11R_-GFP had no effect on the L-LTP.Insets, Representative field EPSPs before and 3 hr after tetanic stimulations are shown. Calibration: 10 msec, 2 mV.C, Comparisons of EPSPs slope 1 hr (a) and 3 hr (b) after tetanic stimulation for induction of either E-LTP or L-LTP, respectively. *p < 0.01 compared with the control slices.

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