Rac1 is a downstream effector of PKCα in structural synaptic plasticity

Abstract

Structural and functional plasticity of dendritic spines is the basis of animal learning. The rapid remodeling of actin cytoskeleton is associated with spine enlargement and shrinkage, which are essential for structural plasticity. The calcium-dependent protein kinase C isoform, PKCα, has been suggested to be critical for this actin-dependent plasticity. However, mechanisms linking PKCα and structural plasticity of spines are unknown. Here, we examine the spatiotemporal activation of actin regulators, including small GTPases Rac1, Cdc42 and Ras, in the presence or absence of PKCα during single-spine structural plasticity. Removal of PKCα expression in the postsynapse attenuated Rac1 activation during structural plasticity without affecting Ras or Cdc42 activity. Moreover, disruption of a PDZ binding domain within PKCα led to impaired Rac1 activation and deficits in structural spine remodeling. These results demonstrate that PKCα positively regulates the activation of Rac1 during structural plasticity.

Figure 1

PKC regulates Rac1 activation during sLTP. (a) Schematic of small GTPase FRET sensors. (b) 2pFLIM images of Rac1 activation in WT slices at indicated time points. Arrowhead represents point of uncaging. Warmer colors indicate higher binding fraction of sensor and higher Rac1 activity. Scale bar, 1 μm. (c) Time courses and quantification of transient (1–3 min) and sustained (10–25 min) spine volume change induced by glutamate uncaging in neurons expressing GFP or in neurons from PKCα WT and KO littermates expressing Rac1 sensor. (d) Time courses and quantification of transient (1.5–3.5 min), sustained (10–25 min) and basal (−8–0 min) Rac1 activation in stimulated spines from WT and PKCα KO littermates. (e) 2pFLIM images of Rac1 activation in PKCα KO slices at indicated time points. Arrowhead represents point of uncaging. Spreading of Rac1 activation in the dendrite were measured in the regions 0 μm from the stimulated spine (red), 1 μm (orange), 2 μm (yellow), 3 μm (brown) and 4 μm (purple). Scale bar, 1 μm. (f) Spatial profile and quantification of spreading Rac1 activation along the dendrite at indicated times and distances from the stimulated spine in WT and PKCα KO littermates. Data are mean ± s.e.m. Grey shading indicates time of uncaging. *P < 0.05, **P < 0.01 two-tailed t-test (c,d) and two-way ANOVA with Sidak’s mutiple comparisons test (f). n (neurons/spines) = 18/22 WT, 19/22 PKCα KO and 5/11 GFP.

Figure 2

PKCα does not regulate Ras or Cdc42 activation during sLTP. (a,c) 2pFLIM images of Ras (a) and Cdc42 (c) activation in PKCα WT and KO neurons at indicated time points. Time courses and quantification of transient (1–3 min) and sustained (10–25 min) spine volume change induced by glutamate uncaging in neurons expressing Ras1 sensor (a) or Cdc42 sensor (c) from PKCα WT and KO littermates. (b,d) Time courses and quantification of transient (1–3 min) and sustained (10–25 min) Ras1 (b) or Cdc42 (d) activation in stimulated spines from WT and PKCα KO littermates. Data are mean ± s.e.m. n(neurons/spines) Ras: n = 8/10 WT, n = 7/8 KO. Cdc42: n = 15/21 WT and n = 7/9 KO *P < 0.05, two-tailed t-test.

Figure 3

PKCα regulates Rac1 activation during sLTP via PDZ binding domain. (a) Primary structure of PKCα showing pseudosubstrate, C1A and C1B domains, C2 domain, kinase domain, C-terminal tail, and PDZ binding motif. (b,c) Time courses and quantification of transient (1–3 min) and sustained (10–25 min) spine volume change (b) and transient and sustained Rac1 activation (c) induced by glutamate uncaging in PKCα KO hippocampal neurons expressing PKCα or PKCα without PDZ domain (PKCα-no PDZ). n = 20/22 PKCα and 14/18 PKCα-no PDZ (neurons/spines). Data are mean ± s.e.m. *P < 0.05, two-tailed t-test. (d) Schematic of potential PKCα regulation of Rac1.