doi: 10.1016/j.neuron.2009.03.013.
Subcellular dynamics of type II PKA in neurons
Affiliations
- PMID:19447092
- PMCID: PMC2702487
- DOI: 10.1016/j.neuron.2009.03.013
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Subcellular dynamics of type II PKA in neurons
Haining Zhong et al. Neuron..
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Abstract
Protein kinase A (PKA) plays multiple roles in neurons. The localization and specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA in neurons and the roles of specific AKAPs are poorly understood. We imaged the distribution of type II PKA in hippocampal and cortical layer 2/3 pyramidal neurons in vitro and in vivo. PKA was concentrated in dendritic shafts compared to the soma, axons, and dendritic spines. This spatial distribution was imposed by the microtubule-binding protein MAP2, indicating that MAP2 is the dominant AKAP in neurons. Following cAMP elevation, catalytic subunits dissociated from the MAP2-tethered regulatory subunits and rapidly became enriched in nearby spines. The spatial gradient of type II PKA between dendritic shafts and spines was critical for the regulation of synaptic strength and long-term potentiation. Therefore, the localization and activity-dependent translocation of type II PKA are important determinants of PKA function.
Figures
(A) Schematic targeting and activation of type II PKA. (B) Representative two-photon images of a CA1 neuron in a cultured hippocampal slice that was co-transfected with RIIβ-mEGFP and DsRed Express. All but B4 are maximum value projections. White box in B3 correspond to the blow-ups in B5 and B6. Arrowheads: B5, a cytoskeletal structure; in B6, axon; B7, presynaptic bouton. Triangles in B6 point to thin dendrites without saturated pixels. Pixel sizes: B1-4, 0.31 μm; B5–7, 0.08 μm. (C) Green/red ratios along the radius away from the center of the soma (seeExperimental Procedures for definition). For the CAT/RIIβ combination (orange), CAT was tagged with mEGFP and RIIβ was untagged. All data were normalized to the value at 12.5 μm from the center of the soma before averaging. n = 5 for mVenus, RIβ and CAT/RIIβ; n = 6 for RIα, RIIα and RIIβ. (D) Green/red ratios along proximal axons, in distal axons, and in boutons. Values are normalized to the G/R of thin basal dendrites. (E) Representative image of CA1 neuron expressing RIIα-mEGFP and DsRed Express. (F) Representative image of CA1 neurons expressing CAT-mEGFP, RIIβ and DsRed Express. Scales of panels E and F are the same as B1 and B5.
(A) Representative images (256×256 pixels, 0.07 μm per pixel) showing spines and their parental dendrites of a CA1 neuron in cultured hippocampal slices that was transfected with CAT-mEGFP, RIIβ and mCherry. Images from individual color channels are shown in grayscale. (B) Representative images (0.10 μm per pixel). Layer 2/3 pyramidal neurons in the somatosensory cortex were transfected byin utero electroporation with CAT-mEGFP and DsRed Express. With (B1, B2) or without (B3) exogenous RIIβ. Arrowheads indicate representative spines. (C) Representative images (0.08 μm per pixel). CA1 pyramidal neurons in cultured hippocampal slices were transfected with DsRed Express and the indicated constructs. (D) Spine enrichment index (SEI) measurements of experiments shown in B, C and E. (E) Representative images (0.07 μm per pixel) indicating that endogenous PKA is enriched in dendritic shafts. Note that the colors for the protein of interest and the cytosol marker are reversed compared to the other experiments. Immunostaining (red) was for PKA catalytic subunits (CAT) or RIIβ on dissociated hippocampal neuronal cultures expressing EGFP (green).
(A) Structure-function studies on type II regulatory subunits. All constructs were tagged with mEGFP. For RIIβ-Δ 2–5-MTBD, the microtubule-binding domain (P272-end) of human MAP2c was tagged on the C-terminus of RIIβ-Δ 2–5. (B, C) Representative images (0.10 μm per pixel) and SEI measurements of CA1 neurons in cultured hippocampal slices prepared from MAP2Δ1–158 −/− mice, their heterozygous littermates and wild-type. RIIβ-mEGFP and DsRed Express were expressed.
(A, B) Representative spine-dendrite images (0.07 μm per pixel) and collective SEI measurements of CA1 neurons expressing CAT-mEGFP, RIIβ and mCherry, before and after bath application of 50μM forskolin and 100 μM IBMX. *, significantly bigger than 0. (C) RIIβ-mEGFP did not move upon forskolin and IBMX activation. (D) Time course and reversibility of the catalytic subunit moving into spines. n = 34. (E) Catalytic subunits expressed in layer 2/3 pyramidal neurons of somatosensory cortex from micein utero electroporated with CAT-mEGFP and DsRed Express also moved to become enriched in spines. With (blue, n = 31) or without (red, n = 41) exogenous RIIβ. (F) Norepinephrine (NE) could initiate the movement of the catalytic subunit into spines. n = 40.
(A) Representative time-lapse images (0.06 μm per pixel). CA1 neurons were expressing CAT-PAGFP, RIIβ and mCherry. Photoactivation was achieved by a nearly instantaneous pulse (~20 ms) of 810nm illumination within the white boxes at 0s. (B) Representative traces of the remaining fluorescence within the activated area. Activation was at the dendrite (left, same as images in panel A) or at the spine (right, the spine above the white box in panel A). (C) Collective decay time constants for dendrites (left) and spines (right). *, significantly smaller.
(A) Averaged uEPSC of all response traces from layer 2/3 pyramidal neurons expressing indicated mEGFP-tagged constructs (solid lines) and untransfected controls from the same slices (dash lines). (B) Averaged peak amplitudes normalized to the same-slice control. *, significantly different. The volumes between the transfected spines and untransfected controls were not significantly different for all pairs.
(A, B and C) Representative traces and averaged, normalized field EPSP amplitudes measured from wild-type, heterozygous and homozygous littermates ofMAP2Δ1–158 mice. LTP were induced with 2 trains of 1s 100 Hz stimulation (blue) or with 900 pulses of 10 Hz stimulation (red and black). For black traces, norepinephrine was applied at -15 to -5 minutes. (D, E) Average potentiation from 50 to 60 minutes by the tetanus LTP induction protocol or the 10 Hz protocol. *, significantly different.
References
- Abel T, Nguyen PV, Barad M, Deuel TA, Kandel ER, Bourtchouladze R. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell. 1997;88:615–626. - PubMed
- Bauman AL, Goehring AS, Scott JD. Orchestration of synaptic plasticity through AKAP signaling complexes. Neuropharmacology. 2004;46:299–310. - PubMed
- Blitzer RD, Connor JH, Brown GP, Wong T, Shenolikar S, Iyengar R, Landau EM. Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP. Science. 1998;280:1940–1942. - PubMed
- Bloodgood BL, Sabatini BL. Neuronal activity regulates diffusion across the neck of dendritic spines. Science. 2005;310:866–869. - PubMed
- Brandon EP, Idzerda RL, McKnight GS. PKA isoforms, neural pathways, and behaviour: making the connection. Curr Opin Neurobiol. 1997;7:397–403. - PubMed
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