PKA/AKAP1 and PP2A/Bβ2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics - PubMed (original) (raw)

PKA/AKAP1 and PP2A/Bβ2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics

Audrey S Dickey et al. J Neurosci. 2011.

Abstract

Mitochondrial shape is determined by fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by dynamin-related protein 1 (Drp1), a large GTPase of the dynamin family that is highly expressed in neurons and regulated by various posttranslational modifications, including phosphorylation. We report here that reversible phosphorylation of Drp1 at a conserved Ser residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase (PP2A/Bβ2) impacts dendrite and synapse development in cultured rat hippocampal neurons. PKA/AKAP1-mediated phosphorylation of Drp1 at Ser656 increased mitochondrial length and dendrite occupancy, enhancing dendritic outgrowth but paradoxically decreasing synapse number and density. Opposing PKA/AKAP1, PP2A/Bβ2-mediated dephosphorylation of Drp1 at Ser656 fragmented and depolarized mitochondria and depleted them from dendrites, stunting dendritic outgrowth but augmenting synapse formation. Raising and lowering intracellular calcium reproduced the effects of dephospho-Drp1 and phospho-Drp1on dendrite and synapse development, respectively, while boosting mitochondrial membrane potential with l-carnitine-fostered dendrite at the expense of synapse formation without altering mitochondrial size or distribution. Thus, outer mitochondrial PKA and PP2A regulate neuronal development by inhibiting and promoting mitochondrial division, respectively. We propose that the bioenergetic state of mitochondria, rather than their localization or shape per se, is the key effector of Drp1, altering calcium homeostasis to modulate neuronal morphology and connectivity.

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Figures

Figure 1.

Figure 1.

PKA/AKAP1 and phospho-Drp1 induce elongation, while PP2A/Bβ2 and dephospho-Drp1 induce fragmentation of mitochondria. A–C, Hippocampal neurons cotransfected at 12–13 DIV with mitochondria-localized GFP, and the indicated constructs were fixed and imaged 4 d later. Representative confocal images. (white: mitochondria) are shown in A. Morphology of dendritic mitochondria is quantified as aspect ratio and form factor in B (means ± SE of 4 experiments, 45+ neurons/condition), and as length in C (means ± SE of three experiments, 36+ neurons/condition). AKAP1 ΔPKA, I310P, L316P mutant; Bβ2M, RR168EE mutant; Ctrl, scrambled shRNA control; ***p < 0.001.

Figure 2.

Figure 2.

PP2A/Bβ2 and dephospho-Drp1 decrease, while PKA/AKAP1 and phospho-Drp1 increase dendritic mitochondria (mito) content. A–D, Hippocampal neurons cotransfected with mCherry, mitochondria-localized GFP, and other constructs were fixed and imaged at 7 (C) or 17 DIV (A, B, D). Representative confocal images of primary dendrites are shown in A. Dendritic mitochondria content (ratio of total mitochondria to total dendrite area) is quantified in C and D, while mitochondria number per unit of dendrite length is shown in B. E, F, Hippocampal neurons transfected with the indicated constructs (Drp1 S656A/S656D plasmids also knock down endogenous Drp1) were fixed at 17 DIV and stained for mitochondria (TOM20) and endosomes (EEA1). E shows representative images of primary dendrites, and F shows total mitochondria and endosome (endo) area over primary dendrite area. Bar graphs show means ± SE of three experiments with 45+ (B, D) and 36+ (C, F) neurons/condition; *p < 0.05, **p < 0.01, ***p < 0.001. Ctrl, Control.

Figure 3.

Figure 3.

PKA/AKAP1 fosters while PP2A/Bβ2 retards dendrite outgrowth. A–E, Hippocampal neurons virally transduced at 0 or 7 DIV were fixed and labeled for MAP2B at 7 (A, E) or 17 DIV (A–D); representative images are shown in A. Dendrite complexity was quantified by Sholl analysis as the number of dendritic intersections with 25 μm-spaced concentric circles centered on the cell body (B, E) and by counting primary dendrites (C) and dendritic branch points per neuron (D). Bar graphs show means ± SE of 3–5 experiments with 36+ neurons/condition; *p < 0.05, **p < 0.01, ***p < 0.001. Ctrl, Control.

Figure 4.

Figure 4.

Mitochondrial fission promotes activity-dependent synapse formation. A–G, HC neurons were virally transduced at 3 DIV and fixed at 17–18 DIV. In G, excitatory neurotransmission was blocked with TTX (1 μ

m

) and APV (100 μ

m

) throughout this period. Synapses were identified by immunolabeling for presynaptic and postsynaptic markers, bassoon/F-actin (A–D) and vGluT1/PSD95 (E, F), and regions of overlap (A, D, E, F) or presynaptic puncta (C, D) were counted by automated image analysis. Numbers of presynaptic, postsynaptic, and overlap puncta were essentially identical for each neuron. Representative confocal images of primary dendrites (A, E) and full dendritic arbors (C) are shown. Synapse density (number per 10 μm primary dendrite) is plotted in B, F, G, while total synapse number (per 150 × 150 μm visual field) is shown in D as means ± SE of 3–4 experiments with 36+ neurons/condition; *p < 0.05, **p < 0.01, ***p < 0.001, compared to control (Ctrl).

Figure 5.

Figure 5.

PP2A/Bβ2 and PKA/AKAP1 act through (de)phosphorylation of Drp1 at Ser656. A–D, Hippocampal neurons transfected at 13 DIV were fixed at 18 DIV and immunolabeled for mitochondria (mito, TOM20; A, B) or glutamatergic synapses (vGluT1 + PSD95, C, D). Representative confocal images of primary dendrites are shown in A and C, while mitochondrial length and synapse density are plotted in B and D, respectively, as means ± SE of three experiments with 36+ neurons/condition; *p < 0.05, **p < 0.01, ***p < 0.001, compared to control (Ctrl); ##p < 0.1, ###p < 0.001, compared to AKAP1/Bβ2 + Drp1.

Figure 6.

Figure 6.

Mitochondrial fission/fusion determines mitochondrial membrane potential. A, B, Hippocampal neurons virally transduced at 12 DIV with GFP or the indicated GFP fusion proteins were labeled with 20 n

m

TMRM and imaged live by confocal microscopy at 18 DIV. FCCP/oligomycin (0.5 μ

m

/2 μ

m

) and

l

-carnitine (1 m

m

) were added 10 min before imaging. A, Representative images of primary dendrites with TMRM fluorescence intensity shown on a pseudocolor scale. B, Relative mitochondrial membrane potential was expressed as the ratio of background-subtracted TMRM fluorescence in mitochondria over the adjacent dendritic cytosol and plotted as means ± SE of 3–6 experiments with 36–65 neurons/condition; *p < 0.05, **p < 0.01, ***p < 0.001 compared to control (Ctrl; GFP) and #p < 0.05, ##p < 0.01 compared to FCCP/oligomycin.

Figure 7.

Figure 7.

Calcium inhibits dendritogenesis. A–D , Hippocampal neurons were cultured in the presence of elevated calcium (2.3 m

m

), the L-type Ca2+ channel agonist Bay K8466 (50 n

m

), or the cell-permeant calcium chelator BAPTA-AM (10 μ

m

) and fixed and stained for MAP2B at 15 or 19 DIV as indicated. Representative epifluorescence images are shown in A and C, while results from Sholl analyses are plotted in B and D as means ± SE of three experiments with 36+ neurons/condition; ***p < 0.001.

Figure 8.

Figure 8.

l

-Carnitine phenocopies mitochondrial fusion effects on dendrite and synapse development. A–H, At 12 DIV, hippocampal neurons were virally transduced with GFP or Bβ2-GFP and treated with or without

l

-carnitine (LC) (1 m

m

) starting at 13 DIV. At 18 DIV, cultures were fixed and stained for mitochondria (mito, TOM20; A --C), dendrites (MAP2B; D, E), and synapses (PSD95, F–H). Shown are representative images (A, D, F) and quantification of mitochondrial length (B), dendritic mitochondria content (C), dendrite complexity (E), synapse number (within a 318 × 318 μm visual field; G), and synapse density (H) plotting; means ± SE of three experiments with 36+ neurons/condition; ***p < 0.001 compared to untreated controls (Ctrl); ###p < 0.001 compared to Bβ2.

Figure 9.

Figure 9.

Model. Reversible phosphorylation of Drp1 at Ser656 by PKA/AKAP1 and PP2A/Bβ2 regulates neuronal development through mitochondrial (mito) membrane potential and calcium sequestration. See Discussion for details.

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