Physiological activation of synaptic Rac>PAK (p-21 activated kinase) signaling is defective in a mouse model of fragile X syndrome

Abstract

The abnormal spine morphology found in fragile X syndrome (FXS) is suggestive of an error in the signaling cascades that organize the actin cytoskeleton. We report here that physiological activation of the small GTPase Rac1 and its effector p-21 activated kinase (PAK), two enzymes critically involved in actin management and functional synaptic plasticity, is impaired at hippocampal synapses in the Fmr1-knock-out (KO) mouse model of FXS. Theta burst afferent stimulation (TBS) caused a marked increase in the number of synapses associated with phosphorylated PAK in adult hippocampal slices from wild-type, but not Fmr1-KO, mice. Stimulation-induced activation of synaptic Rac1 was also absent in the mutants. The polymerization of spine actin that occurs immediately after theta stimulation appeared normal in mutant slices but the newly formed polymers did not properly stabilize, as evidenced by a prolonged vulnerability to a toxin (latrunculin) that disrupts dynamic actin filaments. Latrunculin also reversed long-term potentiation when applied at 10 min post-TBS, a time point at which the potentiation effect is resistant to interference in wild-type slices. We propose that a Rac>PAK signaling pathway needed for rapid stabilization of activity-induced actin filaments, and thus for normal spine morphology and lasting synaptic changes, is defective in FXS.

Figure 1.

The stabilization of spine F-actin and LTP is impaired in Fmr1-KO hippocampus. A, Photomicrographs (left panels) show in situ Alexa 568-phalloidin labeling of F-actin in spine-like structures in field CA1 of hippocampal slices from KO mice that received TBS and then vehicle (+Veh) or latrunculin A (+LatA) treatment 10 min later; slices were harvested 60 min after TBS. Note the abundance of phalloidin labeled F-actin puncta in the +Veh slice and the absence of this labeling in the slice treated with LatA. Inset shows, at higher magnification, the labeling in the field indicated by the box; as shown, dense F-actin labeling is seen in spines (arrow) often in association with lightly labeled dendritic shafts (arrowhead). Scale bar: A, 20 μm; (for inset), 4 μm. Bar graph (at right) shows quantification of F-actin-enriched spines in KO and WT slices following control stimulation (con) or TBS with or without LatA infusion. Note: LatA applied at 10 min after TBS significantly reduced F-actin labeling in Fmr1-KO slices only (*p < 0.05; Tukey's HSD post hoc). B, Plot showing fEPSP slopes recorded from CA1 str. radiatum in Fmr1-KO and WT slices receiving TBS (arrow) and LatA infusion 10 min later. LatA had no effect on LTP in WT slices but abolished it in KO slices. C, LatA infused at 30 min post-TBS had no effect on potentiation in slices from either genotype. Insets show representative traces (overlaid) collected during baseline (1) or 70–90 min after TBS (2) for each genotype in B and C. Calibration: 1 mV, 10 ms.

Figure 2.

Time period for LTP reversal by manipulations acting on adenosine A1 receptors is not prolonged in Fmr1-KO mice. Local applications of adenosine (200 μm; 4 min) or a 3 min train of 5 Hz stimulation (bar), two treatments known to reverse LTP when applied immediately after induction (TBS, arrow), had no lasting effects on potentiation when applied at 10 min after TBS in hippocampal slices (n = 4) prepared from Fmr1-KO mice. Both manipulations caused a transient depression of synaptic responses, as expected for stimulation of adenosine A1 receptors.

Figure 3.

TBS-induced increases in spine pPAK are absent in Fmr1-KOs. A, Photomicrographs show similar punctate immunoreactivity for pPAK and PSD95 (and merged) in CA1 str. radiatum of control WT and Fmr1-KO hippocampal slices. Scale bar, 10 μm. B, Photomicrographs of a single synapse containing pPAK (red) and PSD95 (green) immunoreactivities displayed (top to bottom) in 90° clockwise turns. Scale bar, 1 μm. C, Photomicrographs show pPAK immunolabeling in WT slices harvested 7 min after receiving control stimulation (Con) or TBS. D, Bar graph shows quantification of pPAK-immunopositive (+) spines (double-labeled for PSD95) in str. radiatum of WT and Fmr1-KO slices that received control stimulation or TBS and collected at the indicated postinduction time points; only the WT slices had elevated numbers of pPAK⁺ spines at 7 min post-TBS (*p = 0.014 vs WT control; ⁺p = 0.035 vs Fmr1-KO, 7 min post-TBS group; n > 8 slices/group).

Figure 4.

TBS fails to activate Rac1 in Fmr1-KO hippocampal spines. A, Photomicrographs show immunoreactivity for Rac1-GTP (green) and cofilin (red; spine marker), and merged image, in CA1 str. radiatum of a WT, control hippocampal slice. As shown, activated Rac1 is localized to a subpopulation of cofilin-labeled spines. Scale bar, 5 μm. B, Quantitative analysis shows the effect of TBS on Rac1-GTP⁺ spines in the two genotypes (levels normalized to respective genotype low-frequency stimulation controls; n = 9–11 slices/group). As shown, TBS increased the number of Rac1-GTP⁺ spines in slices from WT but not Fmr1-KO mice. **p < 0.001, one-tailed Student's t test; planned comparison.

Figure 5.

Abnormal PAK3 levels in Fmr1-KO spines. A, Left, Western blots show total levels of PAK1, PAK3, and actin in hippocampal homogenates from Fmr1-KO and WT mice. Right, Quantitative analysis shows no difference in hippocampal PAK levels (immunoreactive band ODs) between genotypes; levels normalized to β-actin (n = 4/group). B, Left, Photomicrographs show PAK3 immunolabeling of spine-like puncta in CA1 str. radiatum of WT and Fmr1-KO mice. Right, Bar graph shows quantification of PAK3⁺ spines (i.e., PAK3⁺ PSD95 double-labeled puncta) and total numbers of PSD95⁺ spines in the CA1 str. radiatum sample field from Fmr1-KO and WT mice (n = 5–8 mice/group). Results are expressed as percentage of mean WT control values; *p < 0.05, Tukey's HSD post hoc, KO vs WT group.

Figure 6.

Defects in physiologically driven actin signaling at hippocampal synapses in Fmr1-KO mice. Observed impairments and their hypothesized causes are represented in a summary diagram of events involved in the production of stable LTP. Three classes of postsynaptic receptors (-Rs) are engaged by theta bursts, two of which drive cytoskeletal modifications. Bound neurotransmitter receptors (glutamate-Rs) initiate events that promote the full activation of these two groups. The modifier-Rs stimulate RhoA, presumably via multiple GTPase-regulatory factors (GRFs), which then initiate a pathway that goes through multiple effectors to trigger actin filament assembly. The modifier-R for BDNF (i.e., TrkB) also facilitates Rac>PAK signaling, which drives unknown effectors to stabilize the newly formed filaments. Adhesion-Rs belonging to the β1 integrin family also drive the RhoA assembly cascade and are assumed from the literature to have potent effects on the Rac, stabilization pathway. Past studies showed that the RhoA-initiated sequence is intact in Fmr1-KO slices; the present findings indicate that physiological activation of Rac and PAK is impaired (slashed lines) in the mutants, and that this is accompanied by a loss of rapid stabilization of both newly formed actin filaments and LTP. Given the results for RhoA, cofilin, and actin polymerization, the defect is not likely to reside in the membrane receptors or their activation. The proposed alternatives are (1) a flaw in the steps leading from the receptors to Rac (dashed arrows) and/or (2) defects in Rac-specific GRFs engaged by the membrane Rs (black ovals). The schematic includes a group of intact Rac-specific GRFs that are not linked to the membrane Rs: these are suggested by the observation that baseline levels of activated Rac and PAK appear normal in Fmr1-KO slices. Finally, myosin IIb is included in the schematic because its regulatory kinase is a target of PAK; disruption of this linkage in the mutants could lead to impaired myosin motor responses to afferent activity, and thus to the abnormalities in PAK distribution described here.