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Inhibition of p21-activated kinase rescues symptoms of fragile X syndrome in mice

Mansuo L. Hayashi^*,, B. S. Shankaranarayana Rao, Jin-Soo Seo, Han-Saem Choi, Bridget M. Dolan^*, Se-Young Choi, Sumantra Chattarji^¶, and Susumu Tonegawa^*,||

*The Picower Institute for Learning and Memory, Howard Hughes Medical Institute, RIKEN–Massachusetts Institute of Technology Neuroscience Research Center, and Departments of Biology and Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore 560029, India; Department of Physiology, College of Dentistry, Seoul National University, Seoul 110-749 Korea; and ^¶National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India

View larger version (42K): [in this window] [in a new window]   Fig. 1. PAK inhibition partially rescues increased density and length of dendritic spines in FMR1 KO mice. (A) Representative dendritic segments of layer II/III pyramidal neurons from WT (n = 20 neurons; two mice), dnPAK TG mice (n = 30 neurons; three mice), FMR1 KO mice (n = 20 neurons; two mice), and double mutant dnPAK TG;FMR1 KO mice (dMT; n = 40 neurons; four mice). (B) On each primary apical dendritic branch, 10 consecutive 10 µm-long dendritic segments were analyzed to quantify spine density. Spine density in dMTs was comparable to WT controls in all dendritic segments except segments 7 and 8 (P > 0.05 in segments 1–6, 9, and 10; P < 0.01 in segments 7 and 8). (C) Mean spine density in dMTs (1.28 ± 0.02) was significantly lower than that in FMR1 KO mice (1.60 ± 0.02; P < 0.001) and significantly higher than that in dnPAK TG mice (1.06 ± 0.01; P < 0.001). ANOVA, P < 0.0001. ***, P < 0.001. (D) As for spine length, FMR1 KO neurons (444 spines) exhibited a significant shift in the overall spine distribution toward spines of longer length compared with WT neurons (406 spines; Kolmogorov-Smirnov test: P < 0.05), whereas dnPAK TG neurons (630 spines) exhibited the opposite shift to shorter spines (P < 0.01). In contrast, spine length distribution in dMT neurons (785 spines) overlapped well with WT neurons and is significantly different from FMR1 KO neurons (P < 0.01).

View larger version (30K): [in this window] [in a new window]   Fig. 2. PAK inhibition rescues reduced cortical LTP in FMR1 KO mice. (A) Input–output curves plotting the changes in field excitatory postsynaptic potential (fEPSP) amplitude and their corresponding presynaptic stimulus intensity in WT (n = 45 slices; 16 mice), dnPAK TG (n = 30 slices; 10 mice), FMR1 KO (n = 57 slices; 19 mice), and dMT mice (n = 24 slices; 8 mice). (B) Cortical LTP induced by TBS was enhanced in dnPAK TG (n = 13 slices; 11 mice), but reduced in FMR1 KO (n = 17 slices; 11 mice), relative to WT controls (n = 17 slices; 11 mice); for responses at 55 min poststimulation, ANOVA, P < 0.05; for both dnPAK TG versus WT and FMR1 KO versus WT, P < 0.04. By contrast, the magnitude of LTP was indistinguishable between dMT slices (n = 13 slices; 9 mice) and WT controls (P > 0.05 for responses at 55 min poststimulation). An overlay of representative field potential traces taken during baseline of recording and at 55 min poststimulation is shown for each genotype.

View larger version (32K): [in this window] [in a new window]   Fig. 3. PAK inhibition partially rescues behavioral abnormalities in FMR1 KO mice. (A–C) Open-field test (WT, n = 10 mice; dnPAK TG, n = 10 mice; FMR1 KO, n = 11 mice; dMT, n = 11 mice). n.s., not statistically different. *, P < 0.05; ***, P < 0.001. (A) FMR1 KO traveled a longer distance compared with WT mice (ANOVA, P < 0.01; WT, 15.29 ± 0.92 m; FMR1 KO, 20.99 ± 1.10 m, P < 0.001). (B) FMR1 KO exhibited a higher number of repetitive behaviors than WT mice (stereotypy counts: ANOVA, P < 0.05; WT, 1,636 ± 119; FMR1 KO, 2,049 ± 125, P < 0.05). (C) FMR1 KO stayed a longer period in the center of the open field than WT mice (ANOVA, P < 0.001; WT, 79.8 ± 8.5 s; FMR1 KO, 143.1 ± 12.0 s, P < 0.001). In all three behaviors, the dMT mice exhibited comparable performance to WT controls (P > 0.05 for all of the following parameters: distance traveled, 17.76 ± 0.91 m; stereotypy counts, 1,756 ± 102; and center time, 108.8 ± 14.6 s). (D–G) Trace fear conditioning task (WT, n = 15 mice; dnPAK TG, n = 12 mice; FMR1 KO, n = 15 mice; dMT, n = 9 mice). n.s., not statistically different. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) On day 1 (conditioning), the four genotypes of mice exhibited comparable amounts of freezing preconditioning (baseline) and postconditioning in all trials. (E) At the 24-h tone test, the four genotypes exhibited comparable amounts of pretone freezing (ANOVA P > 0.05). However, for tone-dependent freezing, FMR1 KO mice and dnPAK TG mice exhibited a significant reduction compared with WT controls (ANOVA for each tone session, P < 0.05; for FMR1 KO versus WT, P < 0.05 for session 1 and P < 0.01 for sessions 2–7; for dnPAK TG versus WT, P > 0.05 for session 1 and P < 0.01 for sessions 2–7). The dMT mice also showed freezing deficits during the first several tone sessions (sessions 1–4) compared with WT controls (P < 0.05). However, with additional tone sessions (sessions 5–7), freezing by dMT mice caught up to that of WT controls (P > 0.05). (F) Average freezing for sessions 1–4. ANOVA P < 0.05. The dMT mice showed freezing deficits compared with WT controls (P < 0.05), but the deficits in dMT mice were less pronounced compared with dnPAK TG (P < 0.01) or FMR1 KO mice (P < 0.01). (G) Average freezing for sessions 5–7. ANOVA P < 0.05. Freezing level in dMT mice was not significantly different from WT controls (P > 0.05), and there were trends in its difference from dnPAK TG (P = 0.12) or FMR1 KO mice (P = 0.07).

View larger version (49K): [in this window] [in a new window]   Fig. 4. PAK1 interacts with FMRP. (A) Immunoprecipitation followed by Western blot analysis. Brain extract was subjected to immunoprecipitation with either rabbit serum (negative control), PAK1 antibody (-PAK1), or -PAK1 plus a blocking peptide. Western blots were probed for either FMRP or PAK1. For Input, 2% of the extract used for a single immunoprecipitation was loaded on the gel. (B) Schematic structure of FMRP, highlighting various functional domains including three RNA-binding motifs (RGG, KH1, and KH2) and the phosphorylation site (S499, represented by a gray asterisk). The constructs used for in vitro binding included full-length (WT), truncated (RGG, S499, and KH), or mutated (I304N) FMRP. RGG refers to the FMRP variant with a deletion of the RGG box at amino acids 526–555. The deleted area in S499 spans amino acids 443–527 and includes the phosphorylation site, S499, as well as putative phosphorylation sites. The isoleucine to asparagine missense mutation in the KH2 domain mimics that previously reported in a human FXS patient (I304N, represented by a black asterisk). The KH deletion mutant lacks both KH domains in tandem corresponding to amino acids 207–425. The numbers refer to the amino acid positions designated by the SwissProt Q06787 entry. Adapted from ref. 38. (C) Characterization of the interaction between PAK1 and various FMRP variants in vitro. In vitro-translated FMRP variants were incubated with GST or GST-PAK1 and glutathione Sepharose beads. The complexes isolated by this method were subjected to SDS/PAGE and Western blotted for FMRP. For Input, 10% of in vitro-translated FMRP sample before the binding reaction was carried out was loaded on the gel.

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